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DNA is a long polymer made from repeating units called nucleotides. The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Å (0.33 nm) long.

The backbone of the DNA strand is made from alternating phosphate and 2-deoxy-ribose, a pentose. The pentoses are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean each strand of DNA has a direction. In a double helix the strands are antiparallel.

The asymmetric ends of DNA strands are called the 5′ and 3′ ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA

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In DNA Replication an entire double-stranded DNA is copied to produce a second, identical DNA double helix. In this process, many different proteins which are clustered together in particular locations in the cell act concertedly. The incoming DNA double helix is split into two single strands and each original single strand becomes half of a new DNA double helix. Because each resulting DNA double helix retains one strand of the original DNA, DNA replication is said to be semi-conservative

These proteins are: •Helicase – which unwinds the DNA double helix into two individual

strands. •Single-strand binding proteins (SSBs), which “coat” the single-

stranded DNA and prevent the DNA strands from reannealing to form double-stranded DNA.

•Primase is an RNA polymerase that synthesizes the short RNA primers needed to start the strand replication process.

•DNA polymerase is an enzyme that strings nucleotides together to form a DNA strand.

•The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication.

•RNAse H removes the RNA primers that previously began the DNA strand synthesis.

•DNA ligase links short stretches of DNA together to create one long continuous DNA strand. 4

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http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

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http://www.youtube.com/watch?v=JRGN35vM4Vw

http://www.youtube.com/watch#!v=AhTKDFxQneY&feature=related

Structure of E. coli helicase RuvA:

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Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis.

Helicases adopt different structures and oligomerization states. Whereas DnaB-like helicases unwind DNA as donut shaped hexamers, other enzymes have been shown to be active as monomers or dimers. The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology (similarities); they all possess common sequence motifs located in the interior of their primary sequence. These are thought to be specifically involved in ATP binding, ATP hydrolysis and translocation on the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.

Based on the presence and the form of helicase motifs, helicases have been separated in 4 superfamilies and 2 smaller families.

Some members of these families are indicated in the next slide, with the organism from which they are extracted, and their function:

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Superfamily I: UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA (Staphylococcus aureus, recombination), Dda (bacteriophage T4, replication initiation), RecD (E. coli, recombinational repair), TraI (F-plasmid, conjugative DNA transfer).

Superfamily II: RecQ (E. coli, DNA repair), eIF4A (Baker's Yeast, RNA translation), WRN (human, DNA repair), NS3[4] (Hepatitis C virus, replication). TRCF (Mfd) (E.coli, transcription-repair coupling).

Superfamily III: LTag (Simian Virus 40, replication), E1 (human papillomavirus, replication), Rep (Adeno-Associated Virus, replication, viral integration, virion packaging).

DnaB-like family: dnaB (E. coli, replication), gp41 (bacteriophage T4, DNA replication),T7gp4 (bacteriophage T7, DNA replication).

Rho-like family: Rho (E. coli, transcription termination).

Note that these superfamilies do not subsume all possible helicases. For example XPB and ERCC2 are helicases not included in any of the above families.

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Science 19 October 2007: Vol. 318. no. 5849, pp. 459 – 463 DOI: 10.1126/science.1147353

Structure of Hexameric DnaB Helicase and Its Complex with a Domain of DnaG PrimaseScott Bailey, William K. Eliason, Thomas A. Steitz*

The complex between the DnaB helicase and the DnaG primase unwinds duplex DNA at the eubacterial replication fork and synthesizes the Okazaki RNA primers. The crystal structures of hexameric DnaB and its complex with the helicase binding domain (HBD) of DnaG reveal that within the hexamer the two domains of DnaB pack with strikingly different symmetries to form a distinct two-layered ring structure. Each of three bound HBDs stabilizes the DnaB hexamer in a conformation that may increase its processivity. Three positive, conserved electrostatic patches on the N-terminal domain of DnaB may also serve as a binding site for DNA and thereby guide the DNA to a DnaG active site.

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STRAND SEPARATIONTo begin the process of DNA replication, the two double helix strands are unwound and separated from each other by the helicase enzyme. The point where the DNA is separated into single strands, and where new DNA will be synthesized, is known as the replication fork.

SSBs, quickly coat the newly exposed single strands and maintain the separated strands during DNA replication. Without the SSBs, the complementary DNA strands could easily snap back together. SSBs bind loosely to the DNA, and are displaced when the polymerase enzymes begin synthesizing the new DNA strands.

NEW STRAND SYNTHESIS

(i)The two single DNA strands can act as templates for the production of two new, complementary DNA strands. Polymerase enzymes can synthesize nucleic acid strands only in the 5’ to 3’ direction, hooking the 5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at the end of the growing nucleic acid chain. Because the chain grows by extension off the 3’ hydroxyl group, strand synthesis is said to proceed in a 5’ to 3’ direction.

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(ii) DNA polymerase can only extend a nucleic acid chain but cannot start one from scratch. To give the DNA polymerase a place to start, an RNA polymerase called Primase first copies a short stretch of the DNA strand. This creates a complementary RNA segment, up to 60 nucleotides long that is called a primer.

Now DNA polymerase can copy the DNA strand. The DNA polymerase starts at the 3’ end of the RNA primer, and, using the original DNA strand as a guide, begins to synthesize a new complementary DNA strand. Two polymerase enzymes are required, one for each parental DNA strand. Due to the antiparallel nature of the DNA strands, however, the polymerase enzymes on the two strands start to move in opposite directions.

One polymerase can remain on its DNA template and copy the DNA in one continuous strand. However, the other polymerase can only copy a short stretch of DNA before it runs into the primer of the previously sequenced fragment. It is therefore forced to repeatedly release the DNA strand and slide further upstream to begin extension from another RNA primer. The sliding clamp helps hold this DNA polymerase onto the DNA as the DNA moves through the replication machinery. The sliding clamp makes the polymerase processive.

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Model for primase structure and function within the replisome. (Inset) Organization of the helicase and primase components of the replisome as observed in the bacteriophage T7 primase-helicase polyprotein. Primase (purple) directly abuts the helicase (gold). The lagging-strand DNA is thought to be threaded through the central channel. (Left and right panels) Models for the orientation of DnaG with respect to DnaB. DNA is shown in blue with synthesized RNA in red. Regions in gray denote the ZBD and DnaB-ID of full-length DnaG whose positions are inferred from the location of the DnaG-RNAP NH2- and COOH-termini. (Left) The primase active site faces away from the central hole of the helicase. ssDNA extruded from the helicase must loop back to reach the primase active site. The direction by which the RNA:DNA hybrid is translocated and ssDNA is extruded are the same (red and blue arrows, respectively). (Right) The DnaG active site faces toward the interior hole of the helicase. Two DnaB protomers have been cut away to show the central hole, where ssDNA from DnaB is guided directly into the DnaG catalytic center for transcription of RNA. The directions of RNA:DNA hybrid translocation and incoming ssDNA are opposed (arrows). Such a model suggests that primer size preferences observed in vitro and in vivo could arise, in part, from steric effects between the primase, helicase, and newly synthesized primer. The directionality of nucleic acid binding to DnaG is indicated as discussed in the text; although a model where DnaG-RNAP binds primer-template in a different configuration cannot be entirely excluded, existing observations agree with the orientation shown.

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In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template.

DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have low processivity. Because the binding of the polymerase to the template is the rate-limiting step in DNA synthesis, the overall rate of DNA replication during the synthesis (or “S”) phase of the cell cycle is dependent on the processivity of the DNA polymerases performing the replication. DNA clamp proteins are integral components of the DNA replication machinery and serve to increase the processivity of their associated polymerases.

Some polymerases add over 50,000 nucleotides to a growing DNA strand before dissociating from the template strand, giving a replication rate of up to 1,000 nucleotides per second.

The continuously synthesized strand is known as the leading strand, while the strand that is synthesized in short pieces is known as the lagging strand. The short stretches of DNA that make up the lagging strand are known as Okazaki fragments.

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THE LAGGING STRAND

Before the lagging-strand DNA exits the “replication factory”, its RNA primers must be removed and the Okazaki fragments must be joined together to create a continuous DNA strand.

The first step is the removal of the RNA primer. RNAse H, which recognizes RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester bonds.

Next, the sequence gap created by RNAse H is then filled in by DNA polymerase which extends the 3’ end of the neighboring Okazaki fragment.

Finally, the Okazaki fragments are joined together by DNA ligase that hooks together the 3’ end of one fragment to the 5’ phosphate group of the neighboring fragment in an ATP- or NAD+-dependent reaction.

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1. The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands and create two replication forks. DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork. Remember that the proteins involved in replication are clustered together and anchored in the cell. Thus, the replication proteins do not travel down the length of the DNA. Instead, the DNA helix is fed through a stationary replication factory like film is fed through a projector. 2. Single-strand binding proteins, or SSBs, coat the single DNA strands to prevent them from snapping back together. SSBs are easily displaced by DNA polymerase. 3. The primase enzyme uses the original DNA sequence as a template to synthesize a short RNA primer. Primers are necessary because DNA polymerase can only extend a nucleotide chain, not start one. 4. DNA polymerase begins to synthesize a new DNA strand by extending an RNA primer in the 5' to 3' direction. Each parental DNA strand is copied by one DNA polymerase. Remember, both template strands move through the replication factory in the same direction, and DNA polymerase can only synthesize DNA from the 5’ end to the 3’ end. Due to these two factors, one of the DNA strands must be made discontinuously in short pieces which are later joined together. 5. As replication proceeds, RNAse H recognizes RNA primers bound to the DNA template and removes the primers by hydrolyzing the RNA. 6. DNA polymerase can then fill in the gap left by RNase H. 7. The DNA replication process is completed when the ligase enzyme joins the short DNA pieces together into one continuous strand.