hUNG2 Is the Major Repair Enzyme for Removal of Uracil from U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with hSMUG1 as a Broad Specificity Backup
hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2–3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup. hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2–3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup. uracil-DNA glycosylase base excision repair AP endonuclease proliferating cell nuclear antigen polymerase enhanced yellow fluorescent protein enhanced cyan fluorescent protein fluorouracil 3,N 4-ethenocytosine single-stranded double-stranded nuclear localization signal hydroxymethyluracil dithiothreitol enhanced green fluorescent protein bovine serum albumin 5-methylcytosine Uracil in DNA can be introduced via two mechanisms, deamination of cytosine and misincorporation of dUMP during replication. Deamination of cytosine has been calculated from measured deamination rates to occur at a rate of 100–500 per human cell/day (1Frederico L.A. Kunkel T.A. Shaw B.R. Biochemistry. 1990; 29: 2532-2537Crossref PubMed Scopus (398) Google Scholar, 2Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4299) Google Scholar) to yield mutagenic U:G mispairs. Uracil may also appear as a consequence of misincorporation of dUMP instead of dTMP during replication, resulting in a U:A base pair. The latter is not miscoding, but may produce cytotoxic and mutagenic AP site intermediates during repair. In organisms containing 5-methylcytosine in their genomes, deamination of 5-methylcytosine furthermore leads to T:G mismatches. All living organisms express uracil-DNA glycosylases (UDGs)1 that prevent cytotoxic and mutagenic effects of the above lesions. UDGs remove uracil (and sometimes other damaged bases or thymine) from the deoxyribose and thus initiate a multistep base excision repair (BER) pathway, eventually restoring the correct DNA sequence. After removal of uracil by an UDG and cleavage of the resulting abasic site by AP endonuclease (APE1/APE2), the BER pathway splits into two branches (reviewed in Ref. 3Dogliotti E. Fortini P. Pascucci B. Parlanti E. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 3-27Crossref PubMed Google Scholar). The presumed major track is the short-patch pathway. It uses the 5′- deoxyribophosphodiesterase activity of DNA polymerase β to cleave 3′ of the abasic site, thus releasing deoxyribose-5-phosphate. Then pol β inserts C or T, depending on the template base. Finally, DNA the by the protein The pathway uses replication and may in replication foci E. T.A. T. PubMed Scopus Google Scholar). pathway pol as as the and polymerase proliferating cell nuclear antigen and the replication C is stimulated by pol which may be in the of The endonuclease the of and DNA the E. Fortini P. Pascucci B. Parlanti E. Prog. Nucleic Acids Res. Mol. 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T. 2001; PubMed Scopus Google Scholar) evidence that a major glycosylase against U:G in also in human and to the hSMUG1 activity against containing U:A and nuclear extracts from and with neutralizing antibodies to hUNG2, or In the of and specificity of the and antibodies antibodies to be and observed with the two enzymes The apparently higher of to hSMUG1 a of the of hSMUG1 to activity DNA as The latter not to a neutralizing nuclear as the of per cell in the present cell to be than hUNG2 is by of not to hSMUG1 by not of the by the also to be a of of the hSMUG1 activity also inhibited at the concentrations of to hUNG2 not to provide the neutralizing antibodies as the present The inhibited nuclear extracts with and of both the of nuclear protein and the of the extracts with inhibited of the activity against both that hUNG2 is the major activity against both misincorporated uracil and deaminated cytosine in nuclear and that of the activity against misincorporated uracil in cell extracts B. 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PubMed Scopus Google and the of hUNG2 in postreplicative removal of misincorporated and from human to initiate BER of uracil in nuclear extracts from and with neutralizing antibodies to to an in BER in of nuclear hUNG2 by BER of the U:A plasmid whereas of hSMUG1 not the of apparently the rate of uracil removal by the two as by excision of uracil from a a to that of the U:A in the BER substrate In the inhibited uracil a U:G plasmid as BER with neutralizing antibodies effect on the of repair because uracil excision from the U:G inhibited by the In BER at of the from short-patch repair of only whereas than from BER not the U:G repair not by excision repair or repair. The latter also by nuclear extracts from in which U:G repair of the plasmid substrate also observed in the of both neutralizing antibodies not Moreover, the short-patch not appear to be on by replication because of from the effect on the to not BER that the of by the neutralizing antibodies with the of the BER with antibodies whereas at the of BER in the that of the that repair of the U:G plasmid by two short-patch BER In the of the short-patch BER In the by a which is not U:G as The of of repair be the nuclear of hUNG2 and hSMUG1 to are for the to the of hUNG2 and hSMUG1 and to their The present strongly that has a broader than postreplicative removal misincorporated and is the major nuclear enzyme for removal of deaminated cytosine in both double-stranded and single-stranded the of hUNG2 with hSMUG1 to remove is to that in higher hSMUG1 of the of as strong to AP broader substrate and the of hSMUG1 in nucleoli but not in replication the activity of hSMUG1 against is nearly as as against that be the substrate for hSMUG1 in is also by the of which is to the by organisms of 5-methylcytosine as a of Nucleic Acids Res. 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Google including the and are not present in the that protein may from The that hUNG2 is a UDG in the of physiological concentrations of may that is an of hUNG2 in occur in both as a consequence of in and in DNA as replication, and because the rate of deamination of cytosine is than higher in ssDNA than in (1Frederico L.A. Kunkel T.A. Shaw B.R. Biochemistry. 1990; 29: 2532-2537Crossref PubMed Scopus (398) Google an for uracil in is by the that the rate in by a rate Google Scholar). removal of deaminated cytosine from ssDNA also be to and of The of for human as a base from a DNA repair enzyme to a enzyme B. PubMed Scopus Google Scholar). hUNG2 two in the E. T.A. T. 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In to may of hUNG2 to single-stranded DNA in of the replication fork. The replication may for and which may be to short-patch repair and repair in the to uracil AP double-stranded DNA the of a a is of in the repair of abasic sites has been B. PubMed Scopus Google Scholar). of deamination of cytosine by is strongly cytotoxic in E. in 2001; PubMed Scopus Google Scholar). of the for and in to enhanced that both and for repair T. PubMed Scopus Google Scholar). The that are for of abasic sites in that may also be the in because is to be We propose that uracil in single-stranded DNA at the replication is by hUNG2 and by or which are both The by and by of We for the cell The cell by of of by of of and of