Article

Interaction of human apurinic endonuclease and DNA polymerase in the base excision repair pathway

Harvard University, Cambridge, Massachusetts, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/1997; 94(14):7166-9. DOI: 10.1073/pnas.94.14.7166
Source: PubMed

ABSTRACT

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polbeta exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

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    • "APEX1 incises the phosphodiester backbone at the 5 -phosphate during repair of an abasic site, leaving a 5 -deoxyribosephosphate (5 -dRP) and 3 -hydroxyl (3 -OH) group [17]. APEX1 enhances binding of DNA Polymerase ␤ (POLB) to DNA and enhances its lyase activity [16] [18], aids in the release of DNA glycosylases from damaged DNA [19], coordinates activities of proteins in long-patch BER [20], and is involved in mitochondrial DNA repair [21]. APEX1 has a redox function, which led to the gene being identified originally as Ref-1 (redox factor-1) [22]. "
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    ABSTRACT: Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Jun 2015
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    • "BER is initiated by the N-methylpurine-DNA glycosylase (MPG, also known as AAG), which recognizes and removes N7-MeG, N3-MeA and N3-MeG (O'Brien and Ellenberger, 2004). The resulting apurinic (AP) site is cleaved by apurinic endonuclease 1 (APE1), leading to the formation of a single-strand break (SSB) (Bennett et al., 1997; Erzberger et al., 1998). The next step of BER is performed by DNA polymerase β (Polß), which inserts a nucleotide and creates 3'-and 5'-ends that can be ligated by DNA ligase III (Srivastava et al., 1998). "
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    ABSTRACT: Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER.
    Full-text · Article · Jun 2014 · The Journal of Cell Biology
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    • "These protein–protein interactions include X-ray cross-complementing factor1 (XRCC1) interactions with pol β, PARP-1, DNA Lig III (22,27) and aprataxin (28), among many others. Protein–protein interactions also have been reported between pol β and the following factors: high mobility group box 1 (HMGB1) (29), DNA Lig I (23), APE1 (30,31), proliferating cell nuclear antigen (PCNA) (32), PARP-1 (30,33), PNK (28), tyrosyl-DNA phosphodiesterase 1 (Tdp1) (34), nei-like-1 (NEIL1) and nei-like-2 (NEIL2) DNA glycosylases (18,35), heat shock protein 70 (36), p53 (37,38), Rad9-Rad1-Hus1 (9-1-1) (39–41) and the adenomatous polyposis coli protein, among others (42). Thus, it appears a wide range of interacting proteins can influence the BER pathway. "
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    ABSTRACT: During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an ‘affinity-capture’ procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3′-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3′-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3′-blocked intermediate.
    Full-text · Article · Oct 2012 · Nucleic Acids Research
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Questions & Answers about this publication

  • Andrei Nikonov added an answer in DNA Binding:
    Why doesn't 100 percent band shift occur in EMSA even when binding saturation has been achieved?

    I am studying DNA binding of a protein using EMSA. The reaction mixture contains 5 nM of DNA substrate while I vary the protein concentration from 2 to 50 nM. I see an increase in band shift with progressive increase in the protein concentration ultimately reaching binding saturation at 50 nM protein. However, it does not lead to 100 percent binding. What might be the reason behind the binding curve not reaching 100%?

    Andrei Nikonov

    I think that you will get many of your answers from the paper attached below:

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      [Show abstract] [Hide abstract]
      ABSTRACT: Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polbeta exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.
      Full-text · Article · Aug 1997 · Proceedings of the National Academy of Sciences