Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Abstract

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).

Figure 1

Substrate and strategy for BER studies. (A) As substrate for the BER assay we used double-stranded covalently closed circular pGEM-3Zf(+) with U/G, U/A, AP-site/G or nicked AP-site/A in the same position. The cleavage sites for restriction enzymes used for mapping of the repair patches are indicated. The shaded region corresponds to the repaired patch. (B) The quality of the substrate in terms of the amount of covalently closed circular plasmid DNA (form I) and nicked circular forms (form II) purified by CsCl/ethidium bromide equilibrium centrifugation. Purity was assessed by running 100 ng of DNA on 1% agarose gel with PstI digested λ DNA as marker. (C) Specificity of anti-UNG-antibodies PU101 and PU1sub. Recombinant UNG2, the mitochondrial form UNG1Δ29 and the catalytic UNG-domain were separated by polyacrylamide gel electrophoresis and subjected to western analysis using either PU101 or PU1sub, as indicated. (D) Structure of the preform of UNG1, UNG1Δ29, UNG2 and the catalytic domain UNGΔ84.

Figure 2

BER carried out by UNG2-ARC. UNG2-ARC was prepared from whole HeLa cell extract and used in BER assays. (A) BER was carried out using U/G substrate or C/G as control and [α-³²P]dCTP as radioactive isotope. After incubation, DNA was purified and digested with restriction enzymes to release short- (SP-BER) and long-patch (LP-BER) repair products as well as total repair products (ligated/unligated products, BamHI/PstI). HMW, high molecular weight band. (B) BER as function of time and input of UNG2-ARC. The indicated amount of beads carrying UNG2-ARC was incubated for specified time periods in BER assay. Short-patch BER products were then analyzed and quantified. (B) Results from a single experiment. (C) Results calculated from four independent experiments with error bars showing SD. Gels were analyzed by phosphorimaging.

Figure 3

Characterization of UNG2-ARC. (A) UNG2-ARC was immunoprecipitated using PU1sub antibodies and non-immunized IgGs as control. Proteins were resolved on a 12% SDS–polyacrylamide gel and silver-stained (upper panel). The lower panel shows BER capacity of the corresponding immunoprecipitates using U/A- and AP/A substrates. (B) Western analysis of UNG2, PCNA, POLδ and XRCC1 in immunoprecipitates. (C) Reduction of UDG-activity in cell extracts by immunoprecipitation. ‘Pre-IP’ indicates UDG-activity prior to immunoprecipitation. The lower panel displays the corresponding western analysis of UNG proteins in cell extracts pre-IP and post-IP. (D) UDG activity of PU1sub- and non-immunized IgG beads. 1×, 7.5 μl input of beads; 2×, 15 μl input of beads.

Figure 4

Specificity in isolation of functional UNG2-ARC and western analysis of proteins present. (A) UNG2-ARC was prepared/attempted to be prepared using PU1sub coated beads, beads coated with non-immune IgG, or pre-immune IgG, which were all incubated with 400 μg HeLa whole cell extracts. The beads were incubated with BER assay mixture containing [α-³²P]dCTP, and U/G, AP/G or AP-nick/G substrate, as indicated. In lane 2, extract was pretreated with 3 U DNase I at 30°C for 30 min prior to UNG2-ARC isolation. Purified DNA was digested for short-patch analysis. (B) Proteins from beads prepared as in (A) were mixed with denaturing loading buffer, heated at 70°C, separated by electrophoresis, blotted and visualized as described in Materials and Methods.

Figure 5

BER and western analysis with UNG^−/− human lymphoblastoid cells. (A) Whole cell extract was prepared from UNG^−/− human lymphoblastoid cells. Whole cell extract (50 μg protein) was used in BER assays with [α-³²P]dCTP and substrates as indicated (lanes 4–6), or ‘UNG2-ARC’ was attempted to be prepared from whole cell extracts using PU1sub antibody, and used for BER assays with substrates as indicated (lanes 1–3). (B) Protein in whole cell extract (lane 2) or from immunoprecipitated ‘UNG2-ARC’ (IP) attempted to be prepared from whole cell extract using PU1sub (lane 1) were subjected to western blot analysis as in Figure 4B.

Figure 6

Roles of UNG2 and SMUG1 in short-patch repair of U/A pairs and U/G mispairs. UNG2-ARC or whole cell extract of HeLa cells was incubated with BER assay mixture for 60 min with [α-³²P]dTTP and U/A (upper panel), or with [α-³²P]dCTP and U/G substrate (lower panel) in the absence or presence of anti-UNG antibody (PU101) or anti-SMUG1antibody (PSM1) or both, as indicated. The diagram represents results from four independent experiments where bars indicate SD.

Figure 7

Lack of functional coupling between the glycosylase step and the subsequent steps in short-patch repair. (A) UNG2-ARC was incubated with BER assay mixture with [α-³²P]dCTP and U/G, AP/G or AP-nick/G as indicated, in the absence or presence of anti-UNG antibody (PU101) and/or anti-SMUG1 antibody (PSM1), and absence or presence of 2 ng recombinant SMUG1 (rec. SMUG1). (B) UNG2-ARC was incubated as in (A), but with [α-³²P]dTTP and U/A, AP/A or AP-nick/A substrate.

Figure 8

Roles of APE1 and XRCC1 in BER. (A) UNG2-ARC was incubated with BER assay mixture in the absence or presence of APE1 and XRCC1 polyclonal antibodies as indicated. (B) Whole HeLa extracts were incubated with XRCC1-antibody linked beads (lanes 2 and 4) or non-immunized serum IgG-linked beads (lanes 1 and 3) for 4 h at 4°C. The beads were removed and the extracts were further incubated with PU1sub-linked beads. The UNG2-ARC thus captured was subsequently used in BER assay.

Figure 9

BER activity in proliferating and non-proliferating cells. UNG2-ARC was isolated from whole extract of exponentially growing HaCaT cells (EC) as well as from whole extract of high-density and growth-arrested HaCaT cells (AC) that did not replicate DNA. (A) Comparison of DNA content distributions as analyzed by flow cytofluorometry analysis of propidium iodide stained HaCaT cells. Grey histogram; exponentially growing proliferating cells, white histogram; growth-arrested non-proliferating cells. Fluorescence intensity is on the linear axis, while the vertical axis indicates the relative number of stained cells. UNG2-ARC was isolated from the extracts and used in BER assays with U/A substrate and [α-³²P]dTTP isotope and incubated for indicated time periods in the absence (lanes 1–6), or presence (lanes 7–8) of neutralizing POLβ antibodies. The repair products were purified and digested with restriction enzymes to release short-patch (B) and long-patch (C) repair products as well as total repair products (ligated/unligated products, BamHI/PstI) (D). Results calculated from two independent experiments, each in duplicate, for BER kinetics analysis of exponentially growing cells relative to growth-arrested HaCaT cells after 15 min incubation and in the presence and absence of anti-POLβ antibodies after 60 min BER incubation (E).

Figure 10

Increased cellular UNG2 protein enhances BER activity in whole cell extract and UNG2-ARC. Exponentially growing HeLa cells carrying a UNG2 expressing construct were either induced with 2 μg/ml of doxycycline for 48 h (+) or mock-induced (−) as control. UNG2-ARC was prepared by incubating 40 μl of PU1sub-linked beads with 100 μg of whole cell extract for each reaction. BER assays with 500 ng of U/A DNA substrate and [α-³²P]dTTP as isotope were carried out using whole cell extracts (40 μg) (lanes 1–6) or UNG2-ARC (lanes 7–18) for the indicated incubation times. The panels below show the results calculated from two independent experiments.