HMGB1 Is a Cofactor in Mammalian Base Excision Repair (original) (raw)
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Toxicology, 2003
The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple pathway requiring only four enzymatic reactions. The key to understand the complete BER process is to elucidate how multiple proteins interact with one another in a coordinated process under specific physiological conditions.
Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity
Nucleic Acids Research, 2016
Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5-deoxyribose phosphate (5-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH 4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.
The EMBO Journal, 2001
The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-speci®c DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase b (Pol b) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a de®ned position. We ®nd that, when repair synthesis proceeds through a Pol b-dependent single nucleotide replacement mechanism, the 5¢-deoxyribosephosphate lyase activity of Pol b is essential for repair of both lesions.
Mammalian base excision repair by DNA polymerases δ and ε
Oncogene, 1998
Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase b (Pol b )-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol b-deleted mouse ®broblasts to separate subfractions containing either Pol d or Pol e. These fractions were then tested for their ability to perform both short-and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a de®ned position. Remarkably, both Pol d and Pol e were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol b. Furthermore, our observations indicated, that either Pol d and/or Pol e participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the ®rst time that Pol d and/or Pol e are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol b in one-gap ®lling reactions.
Nucleic Acids Research, 2009
DNA polymerase h (Pol h) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol h has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol h has intrinsic 5'-deoxyribose phosphate (5'-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol h is ã 300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol h possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH 4 cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol h. These results are consistent with a role of Pol h in BER.
Characterization of the DNA polymerase requirement of human base excision repair
Nucleic Acids Research, 1996
Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase β (Polβ). Purified, recombinant Polβ efficiently substituted for this fraction. Escherichia coli PolI, mammalian Polδ and to a lesser extent Polα and ε also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polβ, which inhibited repair synthesis catalyzed by pure Polβ by ∼90%, only suppressed repair in crude extracts by a maximum of ∼70%. An inhibitor of Polβ, ddCTP, decreased base excision repair in crude extracts by ∼50%, whereas the Polα/δ/ε inhibitor, aphidicolin, reduced the reaction by ∼20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polβ is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.
DNA Repair, 2019
There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or "short-patch" (SP-BER), and "long-patch" BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP-and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SPand LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single-and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ~80% SP-BER. The results show that recombinant Polλ can contribute to both SP-and LP-BER. However, endogenous Polλ, which is present at a level ~50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.