DNA R eplication -III

DNA DNA RReplicationeplication-III-III

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• The origin of replication in E. coli is termed oriC origin of Chromosomal replication

• Important DNA sequences in oriC AT-rich region DnaA boxes

Initiation of Replication

DNA Polymerase III Is the Replicative Polymerase in E. coli

Pol III is responsible for replicating the E. coli chromosome.

The Pol III core is a heterotrimer that contains one each of α, ε, and θ subunits.

The DNA polymerase activity is contained in the α subunit; the γ subunit contains the proofreading 3′→5′ exonuclease.

The Pol III core is just one part of a much larger protein assembly called the Pol III holoenzyme, which replicates both leading and lagging strands.

The Pol III holoenzyme includes two Pol III cores, two ring-shaped β sliding clamps, and one clamp loader.

The clamp loader includes two τ subunits with C-terminal domains that protrude from the clamp loader and bind to the Pol III cores.

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• The Pol III core itself is capable of DNA synthesis at a slow rate, but DNA synthesis by the Pol III holoenzyme is exceedingly rapid, nearly 1 kb/s.

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Many Different Proteins Advance a Replication Fork

DNA Helicase: The two strands of the parental DNA duplex are separated by a class of enzymes known as DNA helicases, which harness the energy of NTP hydrolysis (usually ATP) to drive strand separation

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Topoisomerase: As a helicase separates the parental duplex, the strands must be untwisted.

In E. coli, gyrase, a type II topoisomerase, is the primary replicative topoisomerase

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primases : synthesize short RNA primers specifically for initiating DNA polymerase action.

In E. coli, an RNA primer of 11 to 13 nucleotides is synthesized by the DnaG primase.

RNA primers are needed to initiate each of the thousands of Okazaki fragments on the lagging strand. The leading strand is also initiated by primase at a replication origin.

E. coli DnaG primase must bind the DNA helicase for activity, and this localizes its action to the replication fork

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DNA Polymerase Cannot Initiate new Strands

Pol I and Ligase RNA primers must be removed at the end of each Okazaki fragment and replaced with DNA. This is achieved through the nick translation activity of Pol I.

The nick in the phosphodiester backbone is then sealed by DNA ligase in a reaction that requires ATP (or NAD+ in E. coli).

Ligase acts only on a 5′ terminus of DNA, not on RNA. This specificity ensures that all the RNA at the end of an

Okazaki fragment is removed before the nick is sealed.

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SSB protein maintains the DNA template in the single strand form in order to: • Prevent the dsDNA formation.• Protect the vulnerable ssDNA from nucleases.

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major elements:

• Segments of single-stranded DNA are called template strands.

• Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

• Initiator proteins and DNA helicase binds to the DNA at the replication fork

and untwist the DNA using energy derived from ATP.

• DNA primase binds to helicase producing a complex called a primosome

• Primase synthesizes a short RNA primer of 10-12 nucleotides.

• Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the

RNA primer.

• The RNA primer is removed and replaced with DNA by polymerase I, and the

gap is sealed with DNA ligase.

• Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-

stranded template DNA during the process.

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• The assembly of bacterial replication forks at the origin occurs in steps, starting with the binding of DnaA initiator protein, which melts an A═T-rich region.

• A DnaB helicase is then loaded onto each of the single strands of DNA by the DnaC helicase loader.

• As DNA is unwound by DnaB, DnaG primase synthesizes RNA primers; this is followed by entry of two Pol III holoenzymes to form a bidirectional replication

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Termination of DNA Replication In E. coli, a region located halfway around the chromosome

from oriC contains two clusters of 23 bp sequences called Ter sites.

The arrangement and orientation of Ter sites is such that bidirectional replication forks from oriC can pass through the first set of Ter sites that they encounter, but are blocked by the second set.

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The End Replication Problem in Eukaryotes At the end of a chromosome, after the leading strand has

been completely extended to the last nucleotide, the lagging strand has a single-strand DNA gap that must be primed and filled in.

The problem arises when the RNA primer at the extreme end is removed for replacement with DNA .

There is no 3′ terminus for DNA polymerase to extend from, so this single-strand gap cannot be converted to duplex DNA.

The genetic in formation in the gap will be lost in the next round of replication.

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The eukaryotic cells use telomerase to maintain the integrity of DNA telomere.

The telomerase is composed of Telomerase RNA Telomerase association protein Telomerase reverse transcriptase It is able to synthesize DNA using RNA as the

template.

Telomerase may play important roles is cancer cell biology and in cell aging.

The problem is solved by telomerase

CContinueontinue Telomerase carries its own template

strand in the form of a tightly bound noncoding RNA.

Telomeres at the ends of eukaryotic chromosomes are composed of a repeating unit of a 6-mer DNA sequence (repeating 5′-TTGGGG-3′)

Telomerase extends the 3′ single-stranded DNA end with dNTPs, using its internal RNA molecule as template.

The extended 3′ single strand of DNA is filled in by RNA priming and DNA synthesis. Removal of the RNA primer for this fill-in reaction still leaves a 3′ single-stranded DNA overhang; this end is sequestered by telomere DNA–binding proteins.

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