Translesion synthesis in mammalian cells

DNA damage blocks the progression of the replication fork. In order to circumvent the damaged bases, cells employ specialized low stringency DNA polymerases, which are able to carry out translesion synthesis (TLS) past different types of damage. The five polymerases used in TLS in human cells have different substrate specificities, enabling them to deal with many different types of damaged bases. PCNA plays a central role in recruiting the TLS polymerases and effecting the polymerase switch from replicative to TLS polymerase. When the fork is blocked PCNA gets ubiquitinated. This increases its affinity for the TLS polymerases, which all have novel ubiquitin-binding motifs, thereby facilitating their engagement at the stalled fork to effect TLS

Convergence of Rad6/Rad18 and Fanconi Anemia Tumor Suppressor Pathways upon DNA Damage

Extremely high cancer incidence associated with patients with Fanconi anemia (FA) suggests the importance of the FA signaling pathway in the suppression of non-FA human tumor development. Indeed, we found that an impaired FA signaling pathway substantially contributes to the development of non-FA human tumors. However, the mechanisms underlying the function of the FA pathway remain less understood. Using RNA interfering approach in combining with cell proliferation and reporter assays, we showed that the function of FA signaling pathway is at least partly mediated through coupling with hRad6/hRad18 signaling (HHR6 pathway). We previously reported that FANCD2 monoubiquitination, a hallmark of the FA pathway activation, can be regulated by HHR6. Here we found that hRad18 can also regulate activation of the FA pathway. More importantly, we found that FANCD2 is capable of modulating activity of DNA translesion synthesis polymerase eta, an effector of HHR6 pathway. These results provide novel insights into how the FA pathway is intertwined with HHR6 pathway to maintain chromosomal stability and suppress the development of human cancer, representing an important conceptual advance in the field of FA cancer research

Repair of gaps opposite lesions by homologous recombination in mammalian cells

Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability

hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia

hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase–PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1–MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells

Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors

Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2–3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 >> Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT]10 and [TC]11 microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences

Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

The cell-cycle inhibitor p21CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA

A chromatin-bound kinase, ERK8, protects genomic integrity by inhibiting HDM2-mediated degradation of the DNA clamp PCNA

ERK8 prevents genome instability by blocking the association of the HDM2 E3 ubiquitin ligase with the genome-protecting protein PCNA

Characterization of DNA with an 8-oxoguanine modification

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2′-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2–8 kcal mol−1 over a 16–116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5′ of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.National Institutes of Health (U.S.) (NIH grant R01-CA075576)National Institutes of Health (U.S.) (NIH grant R01-CA055042)National Institutes of Health (U.S.) (NIH grant R01-CA149261)National Institutes of Health (U.S.) (NIH grant P30-ES00002)National Institutes of Health (U.S.) (NIH grant P30-ES02109)National Center for Research Resources (U.S.) (grant number M01RR-01066)National Center for Research Resources (U.S.) (grant number UL1 RR025758, Harvard Clinical and Translational Science Center