Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision, association with PCNA, and stimulation by AP endonuclease.
Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25 bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.
Xia
Liqun
L
Biology Division, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010, USA.
Zheng
Li
L
Lee
Hyun-Wook
HW
Bates
Steven E
SE
Federico
Laura
L
Shen
Binghui
B
O'Connor
Timothy R
TR
D020029:Base Pairing; D002478:Cells, Cultured; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D019271:Hypoxanthine; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase