Human base excision repair complex is physically associated to DNA replication and cell cycle regulatory proteins - PubMed (original) (raw)

Human base excision repair complex is physically associated to DNA replication and cell cycle regulatory proteins

Eleonora Parlanti et al. Nucleic Acids Res. 2007.

Abstract

It has been hypothesized that a replication associated repair pathway operates on base damage and single strand breaks (SSB) at replication forks. In this study, we present the isolation from the nuclei of human cycling cells of a multiprotein complex containing most of the essential components of base excision repair (BER)/SSBR, including APE1, UNG2, XRCC1 and POLbeta, DNA PK, replicative POLalpha, delta and epsilon, DNA ligase 1 and cell cycle regulatory protein cyclin A. Co-immunoprecipitation revealed that in this complex DNA repair proteins are physically associated to cyclin A and to DNA replication proteins including MCM7. This complex is endowed with DNA polymerase and protein kinase activity and is able to perform BER of uracil and AP sites. This finding suggests that a preassembled DNA repair machinery is constitutively active in cycling cells and is ready to be recruited at base damage and breaks occurring at replication forks.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Purification of the multiprotein DNA repair complex. (A) Elution profile of the last purification step (Mono S). POLα (black symbols), POLα/ɛ (open squares) and POLδ/ɛ (open triangles) activities coeluted in the same fractions. (B) Dot blot analysis of the Mono S fractions with specific antibodies showing coelution of cyclin A, POLα, POLδ and LIG1.

Figure 2.

Figure 2.

Protein kinase activity of the multiprotein complex. (A) Phosphorylated polypeptides were revealed upon incubation of the Mono S fraction in the presence of [γ-32P] ATP alone (lane 1), or in combination with histone H1 (lane 2), histone plus p21 (lane 3) or histone plus Olomucine (lane 4). (B) The Mono S fraction was immunoprecipitated with anti-cyclin A antibodies and the immunoprecipitated material was probed with antibodies against cyclin A (lane 1) or anti-phosphoSer/Thr (lane 2).

Figure 3.

Figure 3.

Western blot analysis of the Mono S fraction. The peak fraction 14 from Mono S was analysed by immunoblotting with different antibodies against DNA repair and replication proteins, as described in material and method section. A single asterisk indicates the expected polypeptide, whereas double asterisk mark indicates degradation products.

Figure 4.

Figure 4.

Physical association of DNA replication and repair proteins. (A) Left panel: The Mono S fraction 15 was immunoprecipitated with antibodies against cyclin A, POLβ, POLɛ or anonymous IgGs. Right panel: POLβ null (−/−) or wild type (+/+) mouse embryonic fibroblasts were lysed and the extracts used for immunoprecipitation in the presence of anti-POLβ antibodies. The immunoprecipitated material was then immunoblotted with anti-POLβ and anti-LIG1 antibodies, as indicated. S, supernatant (1:10); IP, immunoprecipitated material. (B) The Mono S fraction 15 was subjected to native gel electrophoresis, followed by immunoblotting analysis with antibodies against POLα, POLβ, POLɛ, LIG1 and XRCC1. As marked by the asterisks, all the antibodies recognized the same high molecular weight band. (C) The Mono S fraction 15 was subjected to gel filtration. Eluted proteins were analysed by dot blot with antibodies against POLα, LIG1, Cyclin A and MCM7. Arrows indicate the corresponding elution points of the molecular weight markers. (D) The gel filtration fraction 10 was analysed by western blot with antibodies against POLα, POLβ, POLδ, DNA PK, cyclin A and MCM7.

Figure 5.

Figure 5.

Mapping of the repair patches at AP site by the multiprotein complex. Top: autoradiograph of a denaturing polyacrylamide gel. Bottom: sequence of the restriction fragments A and B. Repair reactions were performed for 1 h in the presence of radiolabelled dTTP (lanes 1-3-5-7-9) or dCTP (lanes 2-4-6-8-10). Aphidicolin (APH), PCNA and α-POLβ were added as indicated. IS, internal standard.

Figure 6.

Figure 6.

Incision and repair activity at uracil by the multiprotein complex. (A) Incision assay in the absence (lane 1) or presence (lane 2) of the UNG2 inhibitor Ugi. (B) Repair assay in the presence of radiolabelled dTTP (lane 1) or dCTP (lane 2). Bottom: sequence of the restriction fragments A and B. IS, internal standard.

Similar articles

Cited by

References

    1. Frouin I, Montecucco A, Spadari S, Maga G. DNA replication: a complex matter. EMBO Rep. 2003;4:666–670. - PMC - PubMed
    1. Dogliotti E, Fortini P, Pascucci B, Parlanti E. Multiple pathways for DNA base excision repair. The mechanism of switching among multiple BER pathways. Prog. Nucleic Acid Res. Mol. Biol. 2001;68:1–28. - PubMed
    1. Parker A, Gu Y, Mahoney W, Lee SH, Singh KK, Lu AL. Human homolog of the MutY repair protein (hMYH) physically interacts with proteins involved in long patch DNA base excision repair. J. Biol. Chem. 2001;276:5547–5555. - PubMed
    1. Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J. Cell. Sci. 2003;116:3051–3060. - PubMed
    1. Caldecott KW. Mammalian DNA single-strand break repair: an X-ra(y)ted affair. Bioessays. 2001;23:447–455. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources