A Sequence in the N-terminal Region of Human Uracil-DNA Glycosylase with Homology to XPA Interacts with the C-terminal Part of the 34-kDa Subunit of Replication Protein A

Abstract

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast twohybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single-or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair. Uracil-DNA glycosylase (UDG)1 is the first enzyme in base excision repair for removal of uracil from DNA and its main function is probably to remove mutagenic uracil residues resulting from deamination of cytosine in DNA (1). The subsequent steps in the base excision repair pathway include, as the minimal enzymatic requirement in vitro, an apurinic/apyrimidinic endonuclease, a deoxyribophosphodiesterase activity (which may be contributed by DNA polymerase ␤), DNA polymerase ␤, and a DNA ligase (2). In analogy to the complexity of the nucleotide excision repair pathway, base excision repair is likely to be more complex in vivo. This is in fact supported by the finding of an alternative, short patch pathway, requiring proliferating cell nuclear antigen and DNA polymerase ␦ (3, 4). A catalytically fully active form of human UDG has been expressed in Escherichia coli (5) and structure-function relationships determined by site-directed mutagenesis and x-ray crystallography (6). These studies identified this form of human UDG as a one domain structure with a positively charged DNA-binding groove. UDGs are relatively small monomeric enzymes that, at least in vitro, do not require cofactors. However, UDG is preferentially associated with replicating SV40 minichromosomes, indicating a possible interaction with components of the replication machinery (7). The gene encoding the major human UDG, UNG, is transcribed predominantly late in the G 1 -phase, resulting in a 2-3-fold increase in UDG activity early in the S-phase (8). The cell cycle regulation is consistent with the presence of several putative regulatory elements detected in the UNG gene (9), including a putative element for binding of replication protein A (RPA) (10) reported previously in D...

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…Several DNA glycosylases interact with replication related-proteins, namely, PCNA and replication protein A (RPA). MYH and UNG interact with both PCNA and RPA [164][165][166]. We observed stable and functional association between NEIL1 and the WRN protein [167].…”

DNA glycosylases in the base excision repair of DNA

“…DNA strand displacement and excessive gap-filling were observed in cell-free extracts lacking XRCC1, indicating that XRCC1 might serve as a scaffold protein during BER. Moreover, human UDG was recently found to interact with both the 34 kDa subunit and the trimeric form of replication protein A (RPA) [13]. In the alternative BER pathway, a short patch containing the abasic site is excised and replaced by normal nucleotides [14,15].…”

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2

“…DNA replication is a complex process involving numerous proteins yet only four of them, DNA polymerase a subunit I and II, proliferating cell nuclear antigen (PCNA) and DNA polymerase d catalytic subunit have so far been recognized as the products of E2F target genes (Lee et al, 1995;Nishikawa et al, 2000;Pearson et al, 1991;Zhao and Chang, 1997).…”