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Proofreading

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore, it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme

Proofreading

Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.Errors during Replication Are Corrected by the DNA Polymerase EnzymeGenomic DNA is synthesized in...

Long-patch Base Excision Repair

Long-patch Base Excision Repair

Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:

Restarting Stalled Replication Forks

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...

Translesion DNA Polymerases

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Coordination between polymerase beta and FEN1 can modulate CAG repeat expansion.

Yuan Liu¹, Rajendra Prasad¹, William A Beard¹

¹Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.

The Journal of Biological Chemistry

|August 14, 2009

Summary

Oxidized DNA base repair can cause CAG repeat expansion, a key factor in Huntington disease. This study reveals how polymerase beta and FEN1 coordination disruption during base excision repair drives this expansion.

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Published on: September 16, 2019

Area of Science:

Molecular Biology
Genetics
DNA Repair Mechanisms

Background:

8-oxoguanine (8-oxoG), an oxidized DNA base, is linked to neuronal CAG repeat expansion in Huntington disease.
The precise mechanism by which 8-oxoG lesions and their repair contribute to repeat expansion remains largely unknown.

Purpose of the Study:

To elucidate the mechanism by which 8-oxoguanine repair leads to CAG repeat expansion.
To identify the key molecular players involved in this expansion process during DNA repair.

Main Methods:

Utilized wild-type and deficient mouse cell extracts (lacking pol beta and HMGB1) to study 8-oxoG repair.
Investigated the role of polymerase beta (pol beta) and flap endonuclease 1 (FEN1) in CAG repeat expansion during long-patch base excision repair (BER).
Analyzed the impact of High-Mobility Group protein B1 (HMGB1) on DNA repair intermediates and expansion.

Main Results:

Discovered size-limited CAG repeat expansion during 8-oxoG repair in wild-type mouse cell extracts.
Expansion was significantly reduced in extracts deficient in pol beta and HMGB1.
Demonstrated that pol beta-mediated multinucleotide gap-filling synthesis during long-patch BER is a key driver of expansion.
Showed that FEN1 unexpectedly promotes expansion by facilitating ligation of strand slippage-induced hairpins.
Identified the uncoupling of pol beta and FEN1 coordination as critical for expansion.
HMGB1 appears to promote expansion by stimulating APE1 and FEN1 activities.

Conclusions:

This study provides the first evidence that disruption of pol beta and FEN1 coordination during long-patch BER directly causes CAG repeat expansion.
The findings reveal a novel mechanism linking DNA base damage repair to the genetic instability observed in Huntington disease.
Understanding this mechanism opens new avenues for therapeutic strategies targeting DNA repair pathways to prevent repeat expansion.