Human DNA Polymerases λ and β Show Different Efficiencies of Translesion DNA Synthesis past Abasic Sites and Alternative Mechanisms for Frameshift Generation

Blanca, Giuseppina; Villani, Giuseppe; Shevelev, I. A.; Ramadan, Kristijan; Spadari, Silvio; Hübscher, Ulrich; Maga, Giovanni

doi:10.1021/bi049050x

Cited by 64 publications

(80 citation statements)

References 24 publications

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“…For example, overexpression of polymerase ␤ is associated with an increased frequency of Ϫ1 frameshift events (18). Polymerase ␤ is especially prone to bypass the AP site and read the adjacent nucleotide (18,67,68). We have provided evidence that the resulting AP bulges would be quickly processed to give a Ϫ1 frameshift.…”

Section: Human Ber Excises Damaged Nucleotides Present Inmentioning

confidence: 85%

Human Base Excision Repair Creates a Bias Toward −1 Frameshift Mutations

Lyons

O’Brien

2010

Journal of Biological Chemistry

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Frameshift mutations are particularly deleterious to protein function and play a prominent role in carcinogenesis. Most commonly these mutations involve the insertion or omission of a single nucleotide by a DNA polymerase that slips on a damaged or undamaged template. The mismatch DNA repair pathway can repair these nascent polymerase errors. However, overexpression of enzymes of the base excision repair (BER) pathway is known to increase the frequency of frameshift mutations suggesting competition between these pathways. We have examined the fate of DNA containing single nucleotide bulges in human cell extracts and discovered that several deaminated or alkylated nucleotides are efficiently removed by BER. Because single nucleotide bulges are more highly exposed we anticipate that they would be highly susceptible to spontaneous DNA damage. As a model for this, we have shown that chloroacetaldehyde reacts more than 18-fold faster with an A-bulge than with a stable A⅐T base pair to create alkylated DNA adducts that can be removed by alkyladenine DNA glycosylase. Reconstitution of the BER pathway using purified components establishes that bulged DNA is efficiently processed. Single nucleotide deletion is predicted to repair ؉1 frameshift events, but to make ؊1 frameshift events permanent. Therefore, these findings suggest an additional factor contributing to the bias toward deletion mutations.The loss or gain of one or more base pairs is one of the most common types of genetic instability (1). Single nucleotide deletions or insertions occur more frequently than larger deletions or insertions and they cause frameshift mutations if they occur within the open reading frame of a gene. Genomic instability is proposed to be a hallmark of carcinogenesis and the loss or alteration of DNA replication or repair pathways is an early step in the progression of cancer (2, 3). Normally the mismatch DNA repair (MMR) 3 pathway suppresses frameshift mutations by performing replication-coupled DNA repair, but many cancer cells inactivate this pathway through mutations or changes in promoter methylation that reduce expression of one or more proteins in this pathway (4 -6). However, not all cases of increased frameshift frequency can be attributed to defects in MMR (7, 8). Here we consider the possibility that other DNA repair pathways might also play a role in frameshift mutagenesis. Many of the factors that influence the frequency of frameshift mutations are known (9, 10). Streisinger and co-workers (11) proposed that slipping of a primer/template pair within a polymerase active site could lead to a misaligned intermediate that would subsequently be extended to generate a bulged intermediate. Subsequent models for frameshift mutations (1, 6), including the effects of damaged templates and DNA intercalators (12, 13), have incorporated this essential feature (Fig. 1). It is generally assumed that another round of DNA replication is required to make this frameshift event permanent, with one copy that retains the original sequence and ...

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Section: Human Ber Excises Damaged Nucleotides Present Inmentioning

confidence: 85%

Human Base Excision Repair Creates a Bias Toward −1 Frameshift Mutations

Lyons

O’Brien

2010

Journal of Biological Chemistry

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“…The members of the X family of DNA polymerases (DNA Pols ␤, , and in eukaryotes), in particular, can insert nucleotides opposite to certain lesions (22,172). After the Y family, the X family polymerases display the next lowest replication fidelity of the six major DNA polymerase families (119).…”

Section: Other Non-y Family Dna Polymerases Capable Of Tlsmentioning

confidence: 99%

Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance

Waters

Minesinger

Wiltrout

et al. 2009

Microbiol Mol Biol Rev

532

647

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SUMMARY DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol ζ, Pol κ, Pol η, and Pol ι.

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“…An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER) 5 pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2,3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols.…”

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confidence: 99%

“…The major replicative enzyme of eukaryotic cells, DNA pol ␦, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols ␤ and were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol ␦ is the enzyme replicating the lagging strand (6).…”

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The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

Maga

Loon

Crespan

et al. 2009

Journal of Biological Chemistry

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Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol ␦, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol ␤ and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10 4 depurination and 10 2 depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER) 5 pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol ␦, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols ␤ and were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol ␦ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7-9), the action of DNA pol ␦ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol ␣/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol ␣/primase. This results in a more accurate copy synthesized by DNA pol ␦. The intrinsic strand displacement activity of DNA pol ␦, in conjunction with F...

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Human DNA Polymerases λ and β Show Different Efficiencies of Translesion DNA Synthesis past Abasic Sites and Alternative Mechanisms for Frameshift Generation

Cited by 64 publications

References 24 publications

Human Base Excision Repair Creates a Bias Toward −1 Frameshift Mutations

Human Base Excision Repair Creates a Bias Toward −1 Frameshift Mutations

Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance

The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

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