Molecular BiologyReplication: The Machinery

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190018. India.

REPLICATION• THE MACHINERY

• Overall enzymes and proteins needed for synthesis of new DNA are referred to as Replisome

• Comprises of DNA Polymerase together with accessory proteins and enzymes

The main components of the replisome

Component FunctionDNA polymerases

DNA helicaseDNA ligaseDNA primaseDNA topoisomerase

RnaseHSSBP’s

Synthesizes DNA, repairs gaps in lagging strand. Has many forms eachperforming different unique functionUnwinds DNA ahead of replication forkJoins the DNA fragments during lagging strand synthesisSynthesis primer needed to initiate the DNA synthesis by polymeraseChanges the linking number of DNA and hence relaxes the torsional straincaused by helicase activityRemoves RNA primers from the lagging strandKeeps the single strands in linear form

The PolymerasesIrrespective of the source all of DNA polymerases possess some basis properties:

• All catalyze the synthesis of new DNA in 5׳3→׳ direction, with the free 3’ OH group being the point at which the DNA is elongated. Thus all polymerases possess a 5׳3→׳ polymerase activity

• All are template dependent without which they cannot synthesize the new DNA strand. Thus they need to be told when, where & what nucleotide to incorporate for the synthesis of new DNA strand

• All require a primer which provides the free 3׳ OH group to which nucleotide can be added. The free end of primer is called as the primer terminus

• Usually all major polymerases possess 3׳ 5→׳exonucleaseactivity; which is required to check for the correct nucleotide inserted during DNA synthesis - the process called as proofreading

E Coli Polymerases• E. coli possess genes coding for five different polymerases but

three are mainly common - I, II & III

• DNA polymerase III is the principle polymerase and unlike others is a multimeric complex of 18 sub-units of ten types

• {α2ε2θ2τ2β4γ2δδχ́ψ}

• The holoenzyme called as DNA polymerase III* is subassembly of 14 subunits consisting of

• two core polymerase of 8 subunits α2ε2θ2τ2 (each core polymerase is αεθ while τ acts as dimerisation subunit )and

• α has polymerase activity

• ε has proof reading activity

• clamp-loading complex of six subunits γ2δδχ́ψ

• DNA polymerase III* has very low processivity but by binding of dimer of β subunit to each core polymerase, processivity is increased to greater than 500000

E Coli Polymerases

E Coli Polymerases

E Coli PolymerasesComparison of DNA polymerases of E. coli

Pol I Pol II Pol III

Gene

Structure

Polymerization rate

Processivity

׳3→׳5 polymerase׳5→׳3 exonuclease׳3→׳5 exonuclease

polA

Polypeptide

16-20

3-200

YesYesYes

polB

Polypeptide

40

1500

YesYesNo

polC, dnaE, dnaN, dnaQ etc

Multimeric complex

250-1000

>500000

YesYesNo

E Coli Polymerases• DNA polymerase I is unique

polymerase because it possesses an additional 5’→3’ exonucleaseactivity in its N-terminal domain

• This could be separated from the main enzyme by mild protease treatment; remaining large fragment is called as Klenowfragment (68kD) remains with its 5’→3’ polymerase & 3’→5’ exonuclease activity intact

• This polymerase plays role in nick translation

Eukaryotic Polymerases• Eukaryotic cells contain four nuclear DNA polymerases and a

fifth which is responsible for organelle genome replication

• The nuclear enzymes are DNA polymerases α, β, δ and ε. DNA polymerases α and δ are responsible for chromosomal replication

• DNA polymerase α is the first polymerase which recognizes the origin of replication after the activation of the replication complex by cyclins and with its associated primase activity it synthesizes the 7-10nt RNA run on the template DNA and then extends it to about 15nt of DNA

• Then replication factor C causes the “polymerase switching” thereby displacing pol α and loading PCNA on the template DNA near the primer strand following which pol δ binds to PCNA and extends DNA strand

• DNA polymerase δ has a proofreading capability

Eukaryotic Polymerases• PCNA (proliferating cell nuclear antigen) is a cyclin

analogous to the E. coli pol III β subunit in that it acts as a sliding ring to increase enzyme’s processivity

• DNA polymerase δ synthesizes both leading and lagging strands

• The function of DNA polymerase α is to extend the RNA in the lagging strand and provide the template for an accessory factor, replication factor (RF-C) whose role may be analogous to that of the E. coli γ-complex, i.e. to load DNA polymerase and control the processivity of replication on the lagging strand

• The role of DNA polymerase ε unclear

• DNA polymerase β is the smallest of the five enzymes and the one with the lowest fidelity and processivity

• DNA polymerase γ (similar to E. coli pol I) is responsible for the replication of mitochondrial DNA

Eukaryotic Polymerases

Comparison of eukaryotic DNA polymerases

α β γ δ ε

Gene (yeast)LocationSubunits

PrimaseAssociated factors

Processivity׳3→׳5 polymerase׳5→׳3 exonuclease

POL1Nuclear4

YesNone

ModerateYesNo

POL4Nuclear1

NoNone

LowYesNo

MIP1Mitochondrial2

NoNone

HighYesYes

POL3Nuclear2

NoPCNA

High with PCNAYesYes

POL2Nuclear>1

NoNone

HighYesYes

DNA Primases

• They synthesize RNA primers for DNA synthesis

• Primases perform two functions

• Firstly, they initiate leading-strand synthesis at the origin

• Secondly, they facilitate the repetitive initiation of Okazaki fragments during elongation

• In E. coli DNA primase is encoded at the dnaG locus

• For the replication of oriC-type replicons, the primasedepends on the DnaB helicase for efficient priming activity

• Primase & helicase form a functional complex, the primosome

• In eukaryotes, two polypeptides associated with DNApolymerase α possess primase activity

DNA Helicases

• They translocate along single strands of DNA and use energy derived from ATP hydrolysis to break hydrogen bonds and separate duplex molecules

• Are essential during replication

• to provide single-stranded templates

• they are the first components to join the replisome

• They usually translocate in one direction only along DNA and are classified according to their polarity

DNA Topoisomerases

• They regulate the level of supercoiling of DNA molecules

• Are required during replication to relax the torsional strain generated when helicases unwind the duplex

• They also remove knots and resolve catenanes (interlocked circles) which arise during replication and recombination

• They change the linking number of DNA by breaking one or both DNA strands, by using their own tyrosine residue resulting in a temporary attachment of the enzyme to one of the DNA ends via a phosphotyrosine bond

• They are divided into two functional classes:

• Class I topoisomerases

• Class II topoisomerases

DNA Topoisomerases• Class I topoisomerases cleave only one strand and can thus

catenate/de-catenate substrates containing a nick by changing linking number in steps of ±1, by passing the other strand through the break

DNA Topoisomerases

• Class II topoisomerases cleave both strands and therefore can catenate/decatenate covalently closed circles by changing the linking number in steps of ±2, by transferring the other double stranded segment through the break

• Dna Gyrase; a type II enzyme uses the energy of ATP hydrolysis to introduce negative supercoiling into DNA removing positive supercoiling generated during replication

DNA Nucleases• They digest nucleic acids by hydrolyzing phosphodiester

bonds

• Nucleases are deoxyribonucleases (DNases) if their substrate is DNA and ribonucleases (RNases) if their substrate is RNA

• Exonucleases degrade nucleic acids from one end of the molecule

• Many operate in only in the 5’ → 3’ or 3’ →5’ direction, removing nucleotides only from the 5’ or the 3’ end respectively of one strand of a double-stranded nucleic acid

• Endonucleases degrade nucleic acids in the middle of the DNA molecule (Hexameric Sequences)

• Hydrolyse internal phosphodiester bonds and can therefore use a covalently closed circular template as a substrate

SSBP’s• They are accessory replication proteins lacking enzymatic

activity, but required for efficient activity of other enzymes in the replisome

• SSBP’s perform many functions in the cell concerning the stability of single stranded regions of DNA

• In replication, this involves

• stabilizing the melted origin

• sustaining the activity of helicases

• removing secondary structures from the DNA template and

• the inhibition of nuclease activity

• In both E. coli and eukaryotes, the SSBP’s interacts with primaseor components of the primosome to facilitate specific priming activity

DNA Ligases

• They catalyze phosphodiester bond formation between adjacent nucleotides in double-stranded DNA or at nick

• The 5' nucleotide must have-an intact phosphate group and the 3' nucleotide an intact hydroxyl group

• In bacteria, DNA ligases require a NAD cofactor whereas in eukaryotes and archaea, ligases require ATP

• In both cases, the cofactor supplies an adenylate group which becomes covalently linked to the active site of the enzyme

• This group is then transferred to the 5' nucleotide which is subsequently attacked by the 3' hydroxyl of the adjacent nucleotide to form a phosphodiester bond

DNA Ligases

DNA Ligases

• DNA ligases control the final stage in all DNA repair pathways, the sealing of nicks remaining on one strand of a double-stranded DNA

• Mammals appear to possess at least four ligase activities

• DNA ligase I being the principle enzyme involved in lagging-strand repair

Questions?