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Functional Roles of PARP2 in Assembling Protein-Protein Complexes Involved in Base Excision DNA Repair.

ABSTRACT: Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polβ, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD⁺ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.

SUBMITTER: Vasil'eva I

PROVIDER: S-EPMC8124814 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

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Identification of base excision repair protein complexes

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2024-04-24 | MSV000094615 | MassIVE

Base excision repair.

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The roles of base excision repair enzyme OGG1 in gene expression.

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Emerging Roles of DNA Glycosylases and the Base Excision Repair Pathway.

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X-ray repair cross complementing protein 1 in base excision repair.

Project description:X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.

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Direct and indirect roles of RECQL4 in modulating base excision repair capacity.

Project description:RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H(2)O(2)-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H(2)O(2)-induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H(2)O(2), RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase beta, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity.

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Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair.

Project description:BACKGROUND: Neisseria meningitidis, the causative agent of meningococcal disease, is exposed to high levels of reactive oxygen species inside its exclusive human host. The DNA glycosylase Fpg of the base excision repair pathway (BER) is a central player in the correction of oxidative DNA damage. This study aimed at characterizing the meningococcal Fpg and its role in DNA repair. RESULTS: The deduced N. meningitidis Fpg amino acid sequence was highly homologous to other Fpg orthologues, with particularly high conservation of functional domains. As for most N. meningitidis DNA repair genes, the fpg gene contained a DNA uptake sequence mediating efficient transformation of DNA. The recombinant N. meningitidis Fpg protein was over-expressed, purified to homogeneity and assessed for enzymatic activity. N. meningitidis Fpg was found to remove 2,6-diamino-4-hydroxy-5-formamidopyrimidine (faPy) lesions and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8oxoG) opposite of C, T and G and to a lesser extent opposite of A. Moreover, the N. meningitidis fpg single mutant was only slightly affected in terms of an increase in the frequency of phase variation as compared to a mismatch repair mutant. CONCLUSION: Collectively, these findings show that meningococcal Fpg functions are similar to those of prototype Fpg orthologues in other bacterial species.

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