The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10 -100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress. HSP701 is a member of a family of proteins, the transcription of which is stimulated in response to heat shock, oxidation, and other stresses in eukaryotes and prokaryotes (1, 2). Upon association, HSPs alter the folding of some proteins and often enhance their biological functions (3). In general, the chaperone functions of HSPs improve cellular responses to stress. HSP70 is an ATPase and plays a role in the transport of certain proteins within the cell (4). Although HSP70 helps protect cells against oxidative stress, it is unclear how it functions in this role (3).Base excision repair (BER) is a major repair pathway and is responsible for correcting much of the DNA damage caused by ionizing radiation and reactive oxygen species. Much of the core enzyme machinery involved in BER has been described; however, the regulation and coordination of BER with other cellular processes is not well understood. HAP1 (also known as Ape1 and Ref-1) is the first enzyme in the BER pathway that is utilized to repair most if not all of the types of damage acted on by this process. Thus, it is a good candidate for a target of regulation.We previously reported that human HSP70 could bind to HAP1 as determined by affinity chromatography and hydroxyl radical footprinting (5). Here we confirm the association using immunoprecipitation and electrophoretic mobility shift assays. HSP70 also markedly enhanced the specific endonuclease activity of HAP1. This report and our previously reported results (5) are the first indication of a role for HSPs in BER in human cells. EXPERIMENTAL PROCEDURESMaterials-Recombinant human HAP1 (His 6 -tagged) was expressed in Escherichia coli and purified to apparent homogeneity. The HAP1 expression plasmid was a kind gift from Dr. Ian Hickson (University of Oxford, United Kingdom). Purified human uracil-DNA glycosylase, UDG⌬84, a recombinant enzyme lacking 84 amino acids at the N terminus, was a generous gift from Dr. G. Slupphaug (UNIGEN, University of Trondheim, Norway). Recombinant human HSP70 and rabbit antiserum against HSP70 were purchased from StressGen (Victoria, British Columbia, Canada). Rabbit IgG against HAP1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Bovine serum albumin was obtained from Sigma. USB T4 polynucleotide kinase and [␥-32 P] A...

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase beta, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase beta was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.