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

Abstract

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 ...

Differential role of base excision repair proteins in mediating cisplatin cytotoxicity

“…The results reported here establish that the uracil is removed by a UNG dependent process leading to subsequent BER pathway activation (Figure 6). HeLa cell extracts have proven to be great tools for determining the mechanistic aspects of DNA repair as they are highly enriched for DNA repair proteins [30]. Loss of the BER intermediate upon incubation with UGI, a highly specific inhibitor of UNG, has been helpful in establishing the importance of this glycosylase in cisplatin ICL processing.…”

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

“…DNA polymerase IV, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the −4 position [36]. Overexpression of enzymes of the base excision repair pathway is known to increase the frequency of frameshift mutations [37]. Human polymerase kappa uses a classical Streisinger template-slippage mechanism to generate −1 deletions in repetitive sequences, as do the bacterial and archaeal homologues [38,39].…”

Mechanisms of glycosylase induced genomic instability

“…In contrast to MMR competition, the alternative bulge-excision model requires catalytically active glycosylases. We tested this model, which involves excision and deletion of the bulged base to preferentially generate -1 frameshift events, using wild-type and inactive (E125Q) AAG (Fig 8B; [37,54]).…”

“…However, since AAG catalytic activity appeared to be required for mutagenesis, and AAG overexpression remained mutagenic in strains deficient for MMR, additional mechanisms could be operative. Given the differential effects of AAG overexpression on -1 and +1 frameshift rates [13] and the ability of AAG to remove bulged bases leading to deletion by the BER pathway [37], we hypothesized that excision of bulged bases in homopolymers would generate the observed bias. This hypothesis, which we term the bulge-excision model, is differentiated from other models for glycosylase-induced mutagenesis in microsatellites by the fact that it requires a catalytically active glycosylase.…”