reginald h. garrett charles m. grisham chapter 28 dna metabolism: replication, recombination, and...

Reginald H. GarrettCharles M. Grisham

Chapter 28DNA Metabolism: Replication,

Recombination, and Repair

Outline

• The DNA replication process.• Properties of DNA polymerases. • Why are there several DNA polymerases ?• Eucaryotic DNA replication.• Replication of the ends of chromosomes.• Replication of RNA genomes.• Genetic recombination: shuffling genetic

information.• DNA repair.• The molecular basis of mutation.• Proteins as genetic agents.

The Dawn of Molecular Biology

April 25, 1953

• Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

• The mechanism: Strand separation, followed by copying of each strand.

• Each separated strand acts as a template for the synthesis of a new complementary strand.

28.1 DNA Replication

• DNA replication is semiconservative – one of the two original strands is conserved in each progeny molecule. Replication is neither conservative nor dispersive.

• DNA replication is bidirectional – it proceeds in both directions from the starting point using two replication forks.

• Replication requires unwinding of the DNA helix (using a helicase) and relieving stress due to unwinding (using a topoisomerase).

Features of DNA Replication

• DNA replication is semidiscontinuous. • The leading strand copies continuously.• The lagging strand copies in segments

(discontinuous). The lagging strand is formed from Okazaki fragments, which are joined together to form a continuous strand.

• Synthesis of both new strands occurs in the 5' 3' direction using a 3' 5' template

28.1 DNA Replication

Figure 28.1 DNA replication

Three Steps:1. Initiation: Ori site in E.coli = OriC. This is a 245 bp highly conserved sequence.2. Polymerization: Chain elongation in the 5'-3' direction. 3. Termination: The “ter region” is a ~ 350 kbp seq. that is 180o from OriC.

E. Coli OriC site

OriC in E.coli is where replication is initiated.

There are three-13 bp AT rich concensus sequences on 5' end of OriC, (5'-GATCTNTTNTTTT). These AT rich sites have only two H-bonds per bp (weak binding) and separate to form a bubble. AT rich DnaA binding5' =13=13=13======9====9====9====9====9== 3'To the right of the AT rich region are five-9 bp sites that have opposing sequences (5'-TTATCCACA) and bind as many as 10-20 DnaA proteins (the initiation factor) to form a nucleosome-like structure. These five sites bind DnaA and require hydrolysis of ATP to open the replication bubble in the AT rich region.

E. Coli DNA Priming Events

HU a histone-like protein prevents DnaA from binding at sites other than OriC.

When the loop is open DnaB, a helicase, binds at each fork as a complex (DnaB6•DnaC6•ATP6). DnaC is a chaperone for DnaB and DnaT assists forming the pre-priming complex. DnaC leaves when ATP hydrolyzes.

DnaB continues to unwind increasing the bubble size displacing DnaA as it moves. SSB binds to ssDNA in the open bubble to prevent annealing. Topo II binds ahead of the fork to relieve stress.

E. Coli DNA Priming Events

PriA, PriB and PriC enter the bubble along with DnaG (DNA primase). This completes the primosome which makes RNA primers. Topo II and SSB are not part of the primosome. DNA primase does not need a primer to begin synthesis of RNA primers.

The primers (10-30 bp) begin at the center base of any GTT sequence. Only one primer is needed on the leading strand. After this is made, the primosome moves to the lagging strand and a new primer is started about every 1000 bp.

Figure 28.2 Bidirectional Replication

Comparison of labeling during unidirectional versus bidirectional replication.

An autoradiogram of E. coli chromosome replication.

E. Coli Features of Replication

Replication in E. coli

• Circular, ds DNA, 4600 kbp.• Replication is bidirectional. • The double helix must be unwound - by helicases. • Supercoiling must be compensated - by DNA

topoisomerase (gyrase). • Replication is semidiscontinuous. • Leading strand is formed continuously. • Lagging strand is formed from Okazaki Fragments.

E. Coli DNA Replication Events

Two Pol III holoenzymes (DnaE) enter with a few other proteins to complete the replisome. Pol III is an asymmetric dimer that synthesizes DNA from both template strands simultaneously.

Pol III needs a primer to begin synthesis. The leading strand is synthesized continuously and the lagging strand discontinously. Both are synthesized in the 5'-3' direction.

Synthesis is bidirectional occurring at both replication forks.

The Semidiscontinuous Model

The newly synthesized DNA is in red. (a) Shows leading and lagging strand synthesis.

(b) The action of DNA polymerase. Pol III fits around and moves along each template strand.

Lagging Strand Synthesis

The lagging strand makes a loop to enable both Pol III core units to move in the direction of the replication fork.

The lagging strand begins replication at a primer and proceeds until it runs into another Okazaki fragment. At this point the core unit dissociates, the chain shifts to position another primer, the core rebinds and makes another Okazaki piece.

The lagging strand will always be a little behind the leading strand.

28.2 E. Coli DNA Polymerases

DNA Pol I was discovered in 1957 by Arthur Kornberg and his colleagues. Pol I and Pol II are involved in DNA repair. Pol III is the enzyme responsible for replication of the E. coli chromosome.

Figure 28.4 The chain elongation reaction catalyzed by DNA polymerase. The 3'-OH carries out a nucleophilic attack on the α-phosphoryl group of the incoming dNTP.

PPi is released as a product. The subsequent hydrolysis of PPi by pyrophosphatase renders the reaction effectively irreversible.

Chain Elongation in DNA Replication

28.2 Properties of DNA Polymerases

• E. coli cells have several DNA polymerases.• E. coli DNA polymerase I has three active sites.• E. coli DNA polymerase I is its own proofreader

and editor.• E. coli DNA polymerase III holoenzyme

replicates the E. coli chromosome.• A DNA polymerase III holoenzyme sits at each

replication fork.• DNA polymerases do not make DNA denovo.

Properties of DNA Polymerase I (Pol I)

Replication occurs 5' to 3'

• Nucleotides are added at the 3'-end of the strand. • Pol I catalyzes about 20 cycles of polymerization

before the new strand dissociates from template. • 20 cycles constitutes moderate "processivity".• Pol I from E. coli is a 928 amino acid (109 kD)

monomer. • In addition to 5'-3' polymerase, it also has 3'-5'

exonuclease and 5'-3' exonuclease activities.

3'-Exonuclease Activity of Pol I Removes Nucleotides From the 3'-End of the Chain

Why does Pol I have exonuclease activity?

• The 3'-5' exonuclease activity serves a proofreading function.

• It removes incorrectly matched bases, so that the polymerase can try again.

• The newly-formed strand oscillates between the polymerase and 3'-exonuclease sites, adding a base and then checking it.

3'-Exonuclease Activity of Pol I Removes Nucleotides From the 3'-End of the Chain

Figure 28.5 The 3'-exonuclease activity of DNA Pol I of E. coli.

E. Coli Pol I, The Kornberg Enzyme

5’-3’ polymerase..…3’-5’ exonuclease…..5’-3’ exonuclease

adds DNA corrects errors removes primer

928-324 Klenow fragment 323-1

Okazaki fragments

5’---------------------- --------------------- ------------------3’ 3’-------------------------------------------------------------------5’

Layout of Pol I Enzyme

The 5'-Exonuclease Activity of Pol I Conducts Nick Translation

Nick Translation and Klenows....

• 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation".

• Hans Klenow used either subtilisin or trypsin to cleave between residues 323 and 324, separating 5'-exonuclease (on residues 1-323) and the other two activities (on residues 324-928, the so-called "Klenow fragment”).

• This 5'-exonuclease activity plays an important role in primer removal during DNA replication.

More Features of Replication

• DNA Pol III uses an RNA primer. • A special primase forms the required primer. • DNA Pol I excises the primer and synthesizes up

to the next fragment. • DNA ligase seals the "nicks" between Okazaki

fragments. • See Figure 28.8 (slide 29) for a view of

replication fork.

E. Coli DNA Polymerase III (Pol III)

Polymerase III is the workhorse that carries out replication in E. coli

• At least 10 different subunits. • "Core" enzyme has three subunits - , , and .• Alpha subunit is polymerase. • Epsilon subunit is 3'-exonuclease. • Theta subunit is involved in holoenzyme assembly

and ε-subunit stabilization.• The beta subunit dimer forms a ring around DNA • Enormous processivity – 4.6 million bases!

The Composition of E. coli Pol III

E. Coli Pol III is a Dimeric Polymerase

• One unit of polymerase synthesizes the leading strand, and the other synthesizes the lagging strand.

• The template strand is read in the 3'-5' direction, so DNA synthesis proceeds in the 5'-3' direction.

• Lagging strand synthesis requires repeated priming.

• Primase bound to the DnaB helicase carries out this function, periodically forming new RNA primers on the lagging strand.

• All single-stranded regions of DNA are coated with SSB (single-stranding binding protein).

Figure 28.6 DNA polymerase III holoenzyme is a dimeric polymerase.

E. Coli Pol III

E. Coli Pol III is a Dimeric Polymerase

Figure 28.7(a) Ribbon diagram of the β-subunit dimer of the DNA polymerase III holoenzyme on B-DNA, viewed down the axis of the DNA. One monomer of the β-subunit dimer is blue and the other yellow. (b) Space-filling model of the same structure. The hole formed by the β-subunits is large enough to easily accommodate DNA (diameter approximately 2.5 nm) with no steric repulsion. The rest of Pol III associates with this sliding clamp to form the replicative polymerase.

The Replication Fork Formed Around the Polymerase III – DNA Complex

Figure 28.8 General features of a replication fork. The DNA duplex is unwound by the action of helicase and DNA gyrase and the single strands are coated with SSB. Primase periodically primes synthesis on the lagging strand. Each half of the dimeric replicative polymerase is a “core” polymerase bound to its template strand by a β-subunit sliding clamp.

Joining Okazaki Fragments

DNA ligase seals the nicks using NAD+ for energy after Pol I has removed the RNA primer and stopped synthesis at the next Okazaki fragment.

Some organisms use ATP to adenylate the ligase.

E-lys + NAD+ --> AMP-NHlys-E + NMP

5'-p-DNA + AMP-NHlys-E --> AMP-5'P-DNA + E-lys

AMP-5'P-DNA + 3'OH-DNA --> DNA(sealed) + AMP

The E.coli ''ter region'' is a ~350 kbp sequence 180o from Ori C. It contains seven sequences, TerA to TerG, which are binding sites for ''tus'', terminator utilization substance. The Ter sequences are ~ 20 bp long and contain the conserved sequence 5'-GTGTGTTGT-3'. When tus is bound replication stops by blocking the helicase.

G F B C A D E

5' ------------------------------------------------- 3'

Ter region (~4.5 min on clock face)

E.coli Termination

The clockwise replication is stopped at ter B,C,F or G and counterclockwise replication at ter A,D or E. The process is complete when synthesis from the opposite direction reaches the stopped strand.

After the gap has been sealed, the two new DNAs are intertwined (concatenated) and Topo II mediates unraveling these by cleavage and reassembly.

E.coli Termination

DNA Replication in E. coli Requires a Family of Proteins

Some Comparisons in Replication

Procaryotic Eucaryotic

Speed 1000 b/sec 50 b/sec

Okazaki 1000 b 100-200 b

Primer ~30 b ~3-5 b

Ori sites single multiple

Polymerase nuclease no nuclease activity activity

DNA Polymerases Are Immobilized in Replication FactoriesFigure 28.9 The current view of DNA replication has polymerases housed in a replication factory “fixed” to a cellular substructure. This extrudes loops of newly synthesized DNA as parental DNA duplex is fed in from the sides. Parental DNA strands are green; new strands are blue; small circles indicate origin of replication.Fig. 28.8 suggests moving polymerases.

28.3 Multiple DNA Polymerases

• Cells have different DNA polymerases for different purposes. These can be grouped in seven functional families, based on sequence homology.

• Family A includes polymerases involved in DNA repair in bacteria.

• Family B includes the eukaryotic polymerases involved in replication of chromosomal DNA.

• Family C is that of the bacterial chromosomal DNA-replicating enzymes.

• Families X and Y act in DNA repair pathways• Family RT designates retrovirus polymerases and

telomerases. RTs use RNA as a template.

Common Architecture of DNA Polymerases

• Despite sequence variation, the various DNA polymerases follow a common architectural pattern.

• This common structure resembles a right hand, with distinct domains referred to as fingers, palm, and thumb.

• The active site lies in a crevice within the palm domain.

• The fingers act in deoxynucleotide recognition and binding.

• The thumb is responsible for DNA binding.

Common Architecture of DNA Polymerases

Figure 28.10 A structural paradigm for DNA polymerases, bacteriophage RB69 DNA polymerase.

28.4 DNA Replication in Eukaryotic Cells

• DNA replication in eukaryotic cells is similar to that in prokaryotes, but vastly more complex.

• Eukaryotic DNA is organized into chromosomes (with 6 billion base pair distributed among 46 chromosomes).

• The cell cycle controls the timing of DNA replication.

• Eukaryotic cells contain a number of different DNA polymerases (see Table 28.4).

• DNA polymerase δ is the principal DNA replicase, analagous to Pol III in E.coli.

The Eukaryotic Cell Cycle

Figure 28.11 The stages of mitosis and cell division define the M phase. G1 is typically the longest part of the cell cycle; G1 is characterized by rapid growth and metabolic activity. Cells that are quiescent, that is, not growing and dividing (such as neurons), are said to be in G0 phase. The S phase is the time of DNA synthesis. S is followed by G2, a relatively short period of growth when the cell prepares for division (mitosis).

The Cell Cycle Controls the Timing of DNA Replication

• Initiation of replication depends on the origin recognition complex (ORC).

• DNA replication occurs only once per cell cycle.• This is accomplished by dividing initiation of DNA

replication into two steps:1) Licensing of replication origins (late M or

early G1) permits replication by assembling a prereplication complex.

2) The activation of replication at the origins during S phase is through phosphorylation activity of S phase cyclin-dependent kinases.

Terms

• Cyclin protein is so-called due to the cyclic nature of their concentration throughout the cell cycle.

• Cdc proteins related to the cell division cycle.• Cdt1 is a replication factor protein and the Cdt1-

Dbf4 complex is a kinase.• MCM refers to mini-chromosome maintenance

protein that is a helicase.• Sld2 and Sld3 are protein substrates for S-Cdk. • Dpb11 is DNA polymerase B II.

Initiation of Eukaryotic Replication

• Licensing involves highly regulated assembly of initiation control proteins at the ORC to form prereplication complexes (pre-RCs).

• For example, in yeast, ORC binds to origins and then recruits Cdc6, Cdt1, and the MCM proteins.

• MCM proteins are replication licensing factors.• Phosphorylations mediated by S-CDK and Cdc7-

Dbf4 trigger the switch from G1 to S phase.• Phosphorylation of MCM and binding of Cdc45

activates the helicase activity of MCM.• Phosphorylation of Sld2 and Sld3 (these interact

with Dpb11) recruit polymerase to the ORC.

The Initiation of DNA Replication in Eukaryotic Cells

Figure 28.12 Binding of the pre-RC to origins of replication is followed by loading of MCM hexameric helicases, phosphorylation reactions mediated by S-CDK and Cdc7-Dbf4, and binding of the 11-3-2 complex.

The Initiation of DNA Replication in Eukaryotic Cells

Figure 28.12 Phosphorylation of Sld2 and Sld3 leads to the recruitment of DNA polymerase to the replication origins. The two diverging MCM complexes serve as helicases, providing single-strand templates.

Proteins of the Prereplication Complex are AAA+ ATPase Family Members

• Several proteins of the prereplication complex, including Cdc6, the Orc proteins, and the MCM proteins, are AAA+ ATPases (see Chapter 16).

• The binding of ORC and Cdc6 to chromatin in the process of pre-RC assembly is ATP-dependent.

• To establish the pre-RC, the MCM proteins must be in stable association with the origin.

• This stability is achieved following ATP hydrolysis, first by Cdc6 and then by ORC.

• Geminin (a parallel coiled-coil dimer) inhibits DNA replication by preventing the incorporation of MCM complexes into the pre-RC.

Eukaryotic Cells Contain a Number of Different DNA Polymerases

• At least 19 different DNA polymerases have been found in eukaryotic cells so far.

• Multiple polymerases participate in leading- and lagging-strand synthesis, especially α, δ, and ε

• α functions in initiation of nuclear DNA replication.• Polymerase δ is the principal DNA polymerase in

eukaryotic DNA replication.• Through its association with PCNA (proliferating

cell nuclear antigen), polymerase δ carries out highly processive DNA synthesis.

• PCNA is the eukaryotic counterpart of the E. coli β2-sliding clamp and clamps δ to the DNA chain.

Eukaryotic Cells Contain a Number of Different DNA Polymerases

Structure of the Human PCNA Homotrimer

Figure 28.13(a) Ribbon representation of the PCNA trimer with an axial view of a B-form DNA duplex in its center. The molecular mass of each PCNA monomer is 37 kD. (b) Molecular surface of the PCNA trimer-DNA complex.

28.5 Replication of Ends of Chromosomes

• Telomeres are the structures at the ends of eukaryotic chromosomes.

• Telomeres are short (5 to 8 bp) tandemly repeated, G-rich nucleotide sequences that form protective caps 1-12 kbp long on the chromosome ends.

• Vertebrate telomere consensus sequence: TTAGGG.• Telomerase (an RNA-dependent DNA polymerase)

maintains telomere length by restoring telomeres at the 3'-ends of chromosomes.

• Somatic cells, which lack telomerase, inevitably lose bits of their telomeres.

• The telomere theory of aging suggests that cells senesce and die when their telomeres are gone.

Figure 28.14 Telomere replication. (a) In lagging strand replication, short RNA primers (pink) are added and extended by DNA polymerase. (b) Asterisks indicate telomere sequences.

28.5 Replication of Ends of Chromosomes

28.6 Replication of RNA Genomes

• Many viruses have genomes composed of RNA.• DNA is an intermediate in the replication of RNA

viruses.• The viral RNA is as a template for DNA synthesis.• The RNA-directed DNA polymerase is called

reverse transcriptase.• All RNA tumor viruses contain such an enzyme

within their viral particle.• RNA viruses that replicate their RNA via a DNA

intermediate are termed retroviruses.• The primer for reverse transcriptase is a specific

tRNA molecule captured from the host cell.

• Reverse transcriptase transcribes the RNA template into a complementary cDNA strand to form a DNA:RNA hybrid.

• Reverse transcriptase has three enzyme activities:1) RNA-directed DNA polymerase activity.2) RNase H activity (an exonuclease activity that

degrades RNA chains in DNA:RNA hybrids).3) DNA-directed DNA polymerase activity (which

replicates the ssDNA remaining after RNase H degradation of the viral genome, yielding a DNA duplex) which directs the remainder of the viral infection process.


• HIV reverse transcriptase is of great clinical interest because it is the enzyme for AIDS virus replication.

• DNA synthesis by HIV reverse transcriptase is blocked by nucleotide analogs, e.g. AZT and 3TC.

• HIV reverse transcriptase incorporates these analogs into growing DNA chains in place of dTMP (in the case of AZT) or dCMP (in the case of 3TC).

• Once incorporated, these analogs block further chain elongation because the lack a 3'-OH where the next incoming dNTP can be added.

• The high error rate of HIV reverse transcriptase means that the virus is ever changing, which makes it difficult to devise an effective vaccine.


HIV Reverse Transcriptase Inhibitors

Figure 28.15 The structure of AZT (3'-azido-2',3'-dideoxythymidine). This nucleoside is phosphorylated in vitro to form deoxynucleoside-5'-triphosphate substrate analogs for HIV reverse transcriptase.

Figure 28.15 The structure of 3TC (2',3'-dideoxy-3'-thiacytidine). This nucleoside is phosphorylated in vivo to form deoxynucleoside-5'-triphosphate substrate analogs for HIV reverse transcriptase.


28.7 Genetic Recombination: Shuffling Genetic Information • Genetic recombination rearranges genetic

information, creating new associations.• Recombination involving similar DNA sequences is

called homologous recombination.• Transposition is the enzymatic insertion of a

transposon, a mobile segment of DNA. This does not require sequence homology. It is also referred to as nonhomologous recombination.

• The process underlying homologous recombination is termed general recombination.

• General recombination requires the breakage and reunion of DNA strands.

• Meselson and Weigle showed that recombination involves exchange of DNA segments.

Homologous Recombination Proceeds According to the Holliday Model• In 1964, Robin Holliday proposed a model for

homologous recombination.• Two homologous DNA duplexes first juxtapose so

that their sequences are aligned – a process of chromosome pairing called synapsis.

• Recombination starts with introduction of small nicks at homologous sites on the two chromosomes.

• Duplexes partially unwind. The free, single-stranded end of one duplex begins to base-pair with its nearly complementary single-stranded region along the intact strand in the other duplex.

• This process is called strand invasion. Ligation follows, forming a Holliday junction.

Figure 28.18 The + and – signs label strands of like polarity. For example, assume that the two strands running 5' to 3' as read are labeled +; and the two strands running 3' to 5' as read left to right are labeled -. Only strands of like polarity exchange DNA during recombination.

The Holliday Model for Homologous Recombination

The Holliday Model for Homologous Recombination

Figure 28.18 The + and – signs label strands of like polarity. For example, assume that the two strands running 5' to 3' as read are labeled +; and the two strands running 3' to 5' as read left to right are labeled -. Only strands of like polarity exchange DNA during recombination.

The Proteins of General Recombination

• In E. coli, the principal players in recombination are:• The RecBCD enzyme complex, which initiates

recombination.• The RecA protein, which binds single-stranded

DNA, forming a nucleoprotein filament capable of strand invasion and homologous pairing.

• The RuvA, RuvB, and RuvC proteins, which drive branch migration and process the Holliday junction into recombinant products.

• Eukaryotic homologs of these prokaryotic proteins have been identified. So, this basic process of recombination is conserved among organisms.

The Functions of these Proteins

• The RecBCD complex is composed of RecB, RecC, and RecD and has both helicase and nuclease activity. Assembly enhances helicase activity.•RecB: 3' nuclease activity, requires ATP•RecC: sliding clamp•RecD: 5' nuclease activity, requires ATP

• RecBCD initiates recombination by attaching to the end of a DNA duplex and using its helicase function to unwind dsDNA.

• As it unwinds DNA, SSB binds to the single strands.• RecBCD endonuclease activity cleaves ssDNA.

The Functions of these Proteins

• RecBCD continues until meeting a chi (Χ) sequence, 5'-GCTGGTGG-3'. Cleavage occurs on the 3' side of the sequence and the nuclease activity of RecBCD changes to favor the 5' end.

• RecBCD directs binding of RecA to 3'-terminal strand.

• RecA protein, also known as recombinase, is a multifunctional protein for general recombination.

• The nucleoprotein filament formed (RecA-ssDNA) is capable of homologous pairing with a dsDNA (a total of three associated strands).

Figure 28.19 A model of RecBCD-dependent initiation of recombination.RecB RecC RecD

The RecBCD Enzyme Complex Unwinds dsDNA and Cleaves Its Single Strands

RecA Protein Can Bind ssDNA and Then Interact with Duplex DNA• RecA with the 3' end moves along a dsDNA

searching for a region of homology (requires ATP).• RecA then mediates the ATP-dependent DNA

strand invasion leading to formation of a Holliday junction. RecA binds to both ssDNA and dsDNA simultaneously.

• RecA forms a right-handed helical filament, one turn spanning about three nucleotides of DNA.

• The RecA nucleoprotein filament is a scaffold upon which recombination takes place.

• The filament has a deep groove large enough to encompass three strands of DNA.

Strand Separation and Re-pairing into Hybrids Initiates Branch Migration

Figure 28.21 Model for homologous recombination as promoted by RecA enzyme. (a) RecA protein (and SSB) aid strand invasion of the 3'-ssDNA into a homologous DNA duplex, (b) forming a D-loop.(c) The D-loop strand that has been displaced by strand invasion pairs with its complementary strand in the original duplex to form a Holliday junction as strand invasion continues.

RuvA, RuvB, & RuvC Resolve the Holliday Junction to Form Recombination Products• The Holliday junction is processed into

recombination products by RuvA, RuvB, and RuvC.• RuvA and RuvB work together as a Holliday

junction-specific helicase complex that dissociates the RecA filament and catalyzes branch migration.

• Depending on how the strands in the junction are cleaved and resolved, patch or splice recombinant duplexes result.

• RuvC is an endonuclease that resolves Holliday junctions into heteroduplex recombinant products.

• RuvB hexamers are AAA+ -type molecular motors.• The RuvABC system may represent a general

paradigm for DNA manipulation in all cells.

Figure 28.22 Model for the resolution of a Holliday junction in E. coli by the RuvA, RuvB, and RuvC proteins. (b) Model for RuvA/RuvB action. (Left): The RuvA tetramer fits snugly within the Holliday junction point. (Center): Oppositely facing RuvB rings assemble on the heterduplexes, ith DNA passing through their centers. (Right): Binding of RuvC at the junction and strand scission by its nuclease activity.

RuvA, RuvB, & RuvC Resolve the Holliday Junction to Form Recombination Products

Recombination-Dependent Replication Restarts DNA Replication at Stalled Forks• Most replication forks at E. coli initiation sites are

derailed by nicks or more extensive DNA damage.• However, DNA replication can be reinitiated

following replication fork restart.• Repair of stalled forks requires replication,

recombination, and repair enzymes.• Restoration of a fork depends on RecA and

RecBCD.• The E. coli PriA protein binds to D-loops and

coordinates resumption of replication by recruiting DnaB helicase to the loop and reestablishing a fork, complete with two copies of the replicative DNA polymerase.

Transposons are DNA Sequences That Can Move from Place to Place in the Genome• Barbara McClintock first proposed (in 1950) that

activator genes could cause mutations in other genes.

• McClintock’s research showed (surprisingly) that activator genes could move about the genome.

• Her “jumping genes” model was viewed with skepticism at first, but molecular biologists verified her model in the 1970s, and in 1983, she was finally awarded the Nobel Prize in Physiology or Medicine for this remarkable discovery.

• McClintock’s jumping genes are now designated as mobile elements, transposable elements, or simply transposons.

Transposons are DNA Sequences That Can Move from Place to Place in the Genome

Figure 28.23 The typical transposon has inverted nucleotide-sequence repeats at its termini (note error) represented here as the 12-bp sequence ACGTACGTACGT. (a) It acts as a target sequence by creating a staggered cut (b) whose protruding ends are ligated to the transposon (c). Gaps are filled in and ligated (d). Transposon insertion thus generates directed repeats of the target site in the host DNA.

28.8 Repairing DNA

A fundamental difference from RNA, protein or lipid

• All the others can be replaced, but DNA must be preserved.

• Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links.

• The human genome has about 150 genes related to DNA repair. Replication error rate 1:109.

Types of Repair

DNA Repair• Direct repair:

• Photoreactivation - photolyase reverses T=T dimer• Insertase – inserts correct base at AP site•O6–methylguanine methyltransferase (TON = 1)

• Excision repair:•Nucleotide excision - exinuclease• Base excision - glycosidase

• Mismatch repair: nonWatson-Crick base pairs• Recombination repair: Gap in parent• SOS Response: unreadable template

• Chemical reactions that reverse the damage, returning DNA to its proper state, are direct reversal repair systems.

• Single-strand damage repair relies on the intact complementary strand to guide repair.

• Systems repairing single-strand breaks include:•Mismatch repair (MMR).• Base excision repair (BER).•Nucleotide excision repair (NER).

• Double-strand breaks (DSBs) are a particular threat to the genome, because the lost sequence cannot be recovered from the same DNA.

28.8 Repairing DNA

Direct Repair

• Pyrimidine dimers can be repaired by photolyase. Excision repair:• Nucleotide excision repair recognizes and

repairs larger regions of damaged DNA than base excision repair.

• A special endonuclease (excinuclease) nicks both sides of damaged region (~12 residues in E.coli and ~29 in eucaryotes) making a single strand gap.

• Pol I fills the gap and DNA ligase seals the nick.

Thymine Dimer and Photolyase

Figure 28.27 UV irradiation causes dimerization of adjacent thymine bases. A cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers. Photolyase binds at the dimer and uses the energy of visible light to break the cyclobutyl ring, restoring the pyrimidines to their original form.

Base excision repair. Figure 28.28A damaged base (black in next slide) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. AP = apurinic or apyrimidinic (loss of base)Then, an AP endonuclease severs the DNA strand, and an exonuclease removes the AP site and several residues. DNA polymerase I synthesizes the new segment. DNA ligase then repair the gap.Note: AP sites commonly form from spontaneous thermal deglycosylation (~5k b/day humans).

Base Excision Repair

Figure 28.28

Base Excision Repair

Mismatch Repair

• Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it by DNA polymerase-mediated local replication. (CC is most difficult; TG is easiest)

• Methyl-directed pathway (E. coli) is specific for the daughter strand.

• Since methylation occurs post-replication, repair proteins identify methylated strand as parent, remove mismatched bases on the daughter strand and replace them.

Mismatch Repair

• Participating proteins scan DNA looking for mismatches.

• MutS binds at the mismatch. MutH (endonuclease) binds to 6-MeA in a GATC sequence in the parent near the error and cleaves on the 5' side of G.

• MutL links MutS and MutH then a helicase, SSB and exonuclease come in to remove the segment with the mismatch (3' 5').

• Pol III fills the gap and DNA ligase seals.

Figure 28.25 DSB repair through homologous DNA recombination. The orange-red pair of lines symbolizes the double-stranded DNA with a DSB; the black-blue pair represents the sister chromatid. Homologous recombination creates a D-loop (c), and sister chromatid-directed DNA replication restores the information content of the damaged duplex (d-f). Depending on how the Holliday junctions are resolved, the products (g) are either (left) noncrossover or (right) crossover recombinants.

28.8 Homologous Recombination Repair

Figure 28.26 Restarting a stalled replication fork through homologous DNA recombination. A lesion in the DNA is symbolized by a circle; in this case, the lesion is in the leading-strand template (a). Leading-strand synthesis halts because of the lesion (b). Lagging-strand synthesis (red) continues, and the Okazaki fragments are ligated (c). When the leading strand invades the new DNA duplex formed by lagging-strand synthesis, a D-loop is formed and strand exchange occurs. Using the lagging strand as a template, synthesis of the leading strand (black) resumes (d), and the replication fork is reestablished (e).

28.8 Homologous Recombination Repair

Figure 28.24 DSB repair through nonhomo-logous DNA end joining (NHEJ). Ku70/80 binds the ends and recruits a set of proteins that juxtaposes the broken ends. Processing of the ends to generate proper substrates for DNA ligase IV then occurs, followed by DNA-ligase-mediated end joining.Double-strand breaks that arise during the S phase of the cell cycle can be repaired through homologous recombination.

28.8 Nonhomologous DNA End Joining

SOS Response (Error Prone Repair)

• Error prone repair results in inaccurate repair of the daughter strand due to replication through an unreadable template region of the parent.

• The error in the daughter strand compared to the original undamaged parent may or may not be lethal but it is the only path available.

• The option is to not replicate DNA and the cell does not reproduce.

• This process requires expression of the SOS box. Key control proteins are LexA and RecA. SOS proteins can fill in gaps where bases are missing.

28.9 The Molecular Basis of Mutation

• Mutations are permanent changes the sequence of bases in DNA (damage that escapes repair).

• Types:• Substitutions of one base pair for another are

point mutations, these are typically silent.• Frameshifts result from the insertion or

deletion of one or more base pairs, these are most often lethal.

Point Mutations Arise by Inappropriate Base-Pairing

• The two types of point mutations are:• Transitions: replace purine with purine or

pyrimidine with pyrimidine.• Transversions: replace pyrimidine with

purine, or purine with pyrimidine.• Bases rarely mispair (Figure 28.29).• The two types of frameshifts are:

• Insertions (additions): adding an extra nucleotide.

•Deletions: skipping a nucleotide (may result from intercalation of aromatic compound).

Point Mutations Due to Base Mispairings

Figure 28.29(a) An example based on tautomeric properties. (b) A in syn conformation pairing with G. (c) T and C base pairing.

Mutations Can Be Induced by Base Analogs

Figure 28.30 5-Bromouracil usually favors the keto tautomer that mimics the base-pairing properties of thymine, but it frequently shifts to the enol form, whereupon it can base-pair with guanine, causing a T-A to C-G transition.


Figure 28.31(a) 2-Aminopurine normally base-pairs with T but (b) may also pair with cytosine through a single hydrogen bond.


Figure 28.32 Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A-T to G-C transition.

Chemical Mutagens React with the Bases in DNA

Figure 28.33 Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine.A HXG XC U5-MeC T


Figure 28.33 Chemical mutagens. (b) Nitrosamines, organic compounds that react to form nitrous acid, also lead to the oxidative deamination of A and C.


Figure 28.33 Chemical mutagens. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine. The result is a C-G to T-A transition.


Figure 28.33 Chemical mutagens. (d) Alkylation of G residues to give O6-methylguanine, which base-pairs with T.


Figure 28.33 Chemical mutagens (e) Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards.


Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards.

28.10 Proteins As Genetic Agents

• Prions are proteins that can act as genetic agents.• “Prion” is an acronym derived from “proteinaceous

infectious particle”.• Prions are transmissible agents that are apparently

composed only of a protein that has adopted an abnormal conformation.

• They produce fatal degenerative diseases of the central nervous system of mammals and believed to be responsible for the human diseases kuru, Creutzfeld-Jacob disease, and others.

• Prions also cause animal diseases such as scrapie, mad cow disease, and chronic wasting disease (in elk and mule deer).

• PrP, the prion protein, comes in various forms, such as PrPc, the normal cellular form, and PrPsc, the scrapie form.

• These forms are thought to differ only in terms of their secondary and tertiary structure.

• One model suggests that PrPc is dominated by α-helices, whereas PrPsc has both helices and β-strands.

• It is hypothesized that the presence of PrPsc can cause PrPc to adopt the PrPsc conformation.

• Stanley Prusiner received the 1997 Nobel Prize in Physiology or Medicine for his discovery of prions.


Figure 28.34 Speculative models suggest that (a) PrPc is mostly helical, whereas (b) PrPsc has both helices and β-strands.


Gene Rearrangements and Immunology

• Is it possible to generate protein diversity using genetic recombination ?

• Cells active in the immune response are capable of gene rearrangement.

• IgG molecules, the major class of circulating antibodies, are encoded by rearranged genes.

• DNA rearrangements assemble an L-chain gene from 3 separate genes.

• DNA rearrangements assemble an H-chain gene from 4 separate genes.

The Organization of the IgG Molecule

Figure 28.35 Two identical L chains are joined with two identical H chains. Each L chain is held to an H chain via an interchain disufide bond.

Variable regions (purple) = antigen recognition. The antigen binding site is composed of hypervariable residues in the VL and VH regions.

The Collapsed β-Barrel Domain Known As the Immunoglobulin Fold

Figure 28.36 The β-barrel structures for both (a) variable and (b) constant regions are shown. (c) A schematic of the 12 collapsed barrel domains that make up an IgG.

End Chapter 28DNA Metabolism: Replication,

Recombination, and Repair

reginald h. garrett charles m. grisham chapter 28 dna metabolism: replication, recombination, and...

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