Role of DNA Polymerases in Repeat-Mediated Genome Instability

Department of Biology, Tufts University, Medford, MA 02155, USA.
Cell Reports (Impact Factor: 8.36). 11/2012; 2(5). DOI: 10.1016/j.celrep.2012.10.006
Source: PubMed


Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

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Available from: Sergei M Mirkin
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    • "Recent results show that replication in the context of HR repair or HR-dependent fork restart proceeds with less fidelity and more mutations than normal replication, even without the complication of copying DNA repeats (Deem et al., 2011; Hicks et al., 2010; Iraqui et al., 2012). Recently, GAA repeats have been shown to induce mutagenesis up to 8 kb from the repeat site in yeast, which is enhanced in strains with polymerase defects and largely dependent on the Polz TLS polymerase (Saini et al., 2013; Shah et al., 2012; Tang et al., 2013). The evidence indicates that DSBs are formed at the repeat, followed by strand invasion of the sister chromatid, and mutagenic synthesis by Polz during HR repair (Saini et al., 2013). "
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    ABSTRACT: Abstract The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them.
    Full-text · Article · Jan 2015 · Critical Reviews in Biochemistry and Molecular Biology
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    • "It appears that DNA replication [29], repair [30] and recombination [31] may play critical roles in causing GAA repeat instability. It has been found that during DNA replication, expanded GAA repeats resulted in replication fork stalling when GAA repeats were in the lagging strand templates [32]–[35]. This could in turn lead to the formation of hairpin/loop structures on the newly synthesized strand or template strand that further results in GAA repeat expansion and deletion [33]. "
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    ABSTRACT: Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "Many mutations affecting the yeast replicative DNA polymerases cause a Pol ζ-dependent spontaneous mutator phenotype that we refer to as DRIM. We previously demonstrated that the mechanism of DRIM involves impaired replication fork progression in the polymerase mutants (70) that leads to a constitutive monoubiquitination of PCNA (6) and the recruitment of Pol ζ for replication of undamaged DNA (22). To model DRIM in the present study, we use the pol3-Y708A mutation resulting in a single amino acid change in the active site of Pol δ. "
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    ABSTRACT: DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.
    Full-text · Article · Sep 2013 · Nucleic Acids Research
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