Replication_02

• CMG moves physically with replication forks or as the unwindosome moves

Cdc45 consistently co-purifies with the six Mcm and four GINS components in different species, and this CMG complex is known as the replisome progression complex (CMG physically moves with the replication forks) or as the unwindosome (due to its helicase activity in vitro)

Cdc45 is believed to be the rate-limiting factor for replication initiation

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• Cdc45 can physically interact with Mcm and DNA Pol a

Cdc45 is believed to be the rate-limiting factor for replication initiation

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• Cdc45 can physically interact with Mcm and DNA Pol a

• 1 per fork
• 2 per bubble

1

• at the bubble we have two, but at each fork we have one

• prokaryotes: RNA
• eukaryotes: RNA and DNA
• The DNA fragment will be re synthesized when the Okazaki fragment is replaced

The phenomenon of replacing Pol α by DNA pol δ/e is called polymerase switching

DNA pol δ/ε have 3′ → 5′ exonuclease activities and can thus edit the replicated DNA

After the primer is made, replication factor C, RFC (a eukaryotic equivalent of g clamp loader of E. coli) displaces DNA pol ε and attracts proliferating cell nuclear antigen, PCNA (an equivalent of b clamp of E. coli)

PCNA then recruits the main replicative polymerase, DNA pol δ (for lagging strand synthesis) or pol e (for leading strand synthesis), making them processive

On the lagging strand, flap endonuclease 1, FEN-1, removes RNA primers of Okazaki fragments


FEN-1 works in conjunction with DNA Pol δ to replace the primers by nick-translation (equivalent to DNA Pol I of E. coli)

Nicks between individual Okazaki fragments on the lagging strands are sealed by DNA ligase I

The eukaryotic equivalent of the E. coli t-subunit that holds the replisomes of both DNA strands together is unknown

• Topoisomerase I relieves torsional stress in front of the replication fork, Topo II a/b take care of the final untangling

Topoisomerase I relieves torsional stress in front of the replication fork, Topo II a/b take care of the final untangling

• High fidelity replicases
• leading strand

• High fidelity replicases
• lagging strand

• high fidelity repair
• base excision repair

• nuclear replication
• high fidelity replication

The ends of eukaryotic chromosomes are protected by special DNA structures called telomeres

Telomeres are repetitive dsDNA elements composed of tandems of short G-rich regions

TTAGGG

The G-rich strand of the telomere is synthesized by the enzyme called telomerase

Telomerase is a ribonucleoprotein complex that contains a short RNA template and a reverse transcriptase that catalyzes telomere synthesis

Each molecule of telomerase contains two copies of TERC (or telomerase RNA component), two copies of TERT (or telomerase reverse transcriptase), and one copy of Dkc1 (or dyskerin), a protein that stabilizes the telomerase complex

Telomerase makes the G-rich strand by extending the protruding 3’-end of the DNA and using TERC as a template

• Ordinary RNA-primed DNA synthesis then takes place on the recessed strand to make the complementary C-rich strand

The function of telomeric proteins is also necessary for the efficient replication of telomeric regions

The repetitive and GC-rich telomeres are known to form higher-order DNA secondary structures, such as G-quadruplexes


G-quadruplexes are believed to interfere with replication fork progression as well as with telomerase activity

The function of telomeric proteins is required for resolving these problems

For example, targeted deletion of the Shelterin component TRF1 in mice leads to stalled replication forks

A hexameric protein complex, known as Shelterin, assists in T-loop formation and protects telomeric DNA

Telomere shortening provides an explanation for a phenomenon called the Hayflick limit

In 1962 Leonard Hayflick discovered that cultured human cells do not divide indefinitely but after a fixed number of cell divisions senesce and die (due to telomere shortening!)

- 5’-3’ DNA polymerase

- 5’-3’ exonuclease

- 3’-5’ exonuclease

• DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication.
• The main function of the third polymerase, Pol III, is duplication of the chromosomal DNA while other DNA polymerases are involved mostly in DNA repair and translesion DNA synthesis. Together with a DNA helicase and a primase, Pol III HE participates in the replicative apparatus that acts at the replication fork.

• Enzyme
• RNA Primers
• Enables DNA Pol III to make DNA

• Reads 3' to 5'
• Writes 5' to 3'

3' OH group of RNA Primer

Replicates mitochondrial DNA