MOLECULAR MECHANISMS OF DNA REPAIR
IN EUKARYOTES

DNA repair is a cellular mechanism to correct damage to DNA before it becomes fixed as a mutation or chromosomal aberration, which may lead to deleterious results such as cell death or tumorigenesis. A number of anticancer drugs and radiation treatments are designed to induce DNA damage in tumor cells. Therefore, understanding the molecular mechanism of DNA repair is important for reducing the risk of cancer as well as developing more effective cancer therapies.

The research in our laboratory focuses on base excision repair, a mechanism that repairs modified bases and baseless lesions (apurinic/apyrimidinic [AP] sites). These lesions occur spontaneously through normal cellular metabolism or are induced by several carcinogens, anticancer drugs, and ionizing radiation. A typical base excision repair reaction of a modified base consists of five sequential steps: 1) removal of the modified base by a specific DNA-N-glycosylase to leave an AP site, 2) incision of the AP site at its 5' side by AP endonuclease, 3) DNA synthesis at the incised site, 4) excision of a deoxyribose phosphate (dRP) group from the 5'incised AP site, and 5) sealing by DNA ligase. Thus, AP sites are common intermediates in this repair mechanism. Studies of AP site repair with in vitro systems that carry out steps 2 through 5 have revealed that base excision repair can proceed by two alternative pathways: the DNA polymerase b (pol b)-dependent pathway and the proliferating cell nuclear antigen (PCNA)-dependent pathway.

A cyclin-dependent kinase (cdk) inhibitor, p21, can bind to PCNA and consequently modify its activities. To understand the functions of p21 in DNA repair, we examined the effects of p21 on base excision repair in vitro and in vivo.

We found that, when added to in vitro reconstituted systems for AP site repair, p21 was able to inhibit the PCNA-dependent pathway, but did not interfere with the pol bdependent pathway. Analysis of intermediate products accumulated during the p21-inhibited repair reaction indicated that both DNA synthesis and AP site excision by FEN1 were suppressed by p21. Gel filtration analysis of DNA-loaded PCNA revealed that loading of PCNA on circular DNA containing an AP site was blocked by p21, and that the preloaded PCNA was relatively resistant to p21. These results suggest that p21 can suppress the PCNA-dependent base excision repair by blocking the loading of PCNA on DNA.

To investigate the effects of p21 on in vivo repair, we transiently transfected mammalian cells with a p21-overexpressing vector and measured the repair activity of H₂O₂-induced damage by the alkali-comet assay (single cell gel electrophoresis assay). Overexpression of the wild-type p21 resulted in a slower rate of oxidative damage repair compared to mock-transfection. However, overexpression of a point-mutant p21 that has lost ability for interaction with PCNA also suppressed the repair activity to some extent. These results suggest that p21 may suppress base excision repair in vivo not only through interaction with PCNA but also through interaction with cdks and/or cyclins.

Pol b catalyzes excision of a dRP group from a 5'-incised AP site by Schiff base formation between the aldehyde group at C1' of the AP site and the Lys-72 residue followed by b-elimination. The amino-terminal 8-kDa domain of this enzyme is sufficient for this reaction. To understand the dRP excision mechanism at the semi-atomic level, we have introduced a series of site-directed mutations into the 8-kDa domain and analyzed their properties. This year, we focused on two planar residues, His-34 and Tyr-39.

A report of a crystal structure of nicked DNA/ pol b complex revealed that His-34 may be in a stacking position with the base opposite to the 5'-terminal nucleotide at the nicked site. This configuration suggests that the planar structure of His-34 may be important for precise positioning of the 8-kDa domain to the 5'-incised AP site. To test this hypothesis, we replaced this residue with Ala (A), Gln (Q), Phe (F) or Tyr (Y), and measured dRP excision activities of these mutants. H34F and H34Y mutants showed activities similar to the wild type protein, whereas H34Q and H34A significantly reduced dRP excision activity. This result indicates that an amino acid residue with a planar structure can substitute for His-34 without losing the activity.

Spatial proximity of Tyr-39 to Lys-72, the catalytic center, also suggests involvement of Tyr39 in precise positioning of the 8-kDa domain to the 5'-incised AP site. Thus we introduced Ala (A), Cys (C), His (H), Phe (F), and Ser (S) into the 39 position. In this case, however, all the mutants lost most of the dRP excision activity. Even the Y39F mutant retained only a partial activity. This result indicates that Tyr39 is essential not only for its planar structure but also for its hydroxy group. We currently propose two models on the Tyr-39 function, involvement in either protein folding or proton transfer for Lys72 deprotonation, and plan to test them.

BIADE, S., SOBOL, R. W., WILSON, S. H., MATSUMOTO, Y. Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA. J. Biol. Chem. 273:898-902, 1998.

FENG, J., CRASTO, C. J., MATSUMOTO, Y. Deoxyribose phosphate excision by the N-terminal domain of the polymerase b: the mechanism revisited. Biochemistry 37:9605-9611, 1998.

GARY, R., KIM, K., CORNELIUS, H. L., PARK, M. S., MATSUMOTO, Y. PCNA facilitates excision in long-patch base excision repair. J. Biol. Chem. (in press).

KIM, K. BIADE, S., MATSUMOTO, Y. Involvement of FEN1 in base excision DNA repair. J. Biol. Chem. 273:8842-8848, 1998.

MATSUDA, T., TERASHIMA, I., MATSUMOTO, Y., YABUSHITA, H., MATSUI, S., SHIBUTANI, S. Effective utilization of N2-ethyl-2'-deoxyguanosine triphosphate during DNA synthesis catalyzed by mammalian replicative DNA polymerase. Biochemistry (in press).

MATSUMOTO, Y., KIM, K., KATZ, D. S., FENG, J. Catalytic center of DNA polymerase b for excision of deoxyribose phosphate groups. Biochemistry 37:6456-6464, 1998.

MATSUMOTO, Y. Base excision repair assay using Xenopus laevis oocyte extracts. In Methods in Molecular Biology, Vol 113: DNA Repair Protocols: Eukaryotic Systems, edited by D.S. Henderson. Humana Press, Totowa, NJ, pp. 289-300, 1999.

^§   Fox Chase researcher

^a   D.S. Katz: Present address-The Agnes Irwin School, Rosemont, PA 19010

^b   J. Hurwitz: Memorial Sloan-Kettering Cancer Center, New York, NY 10021

^c   M. Park: Los Alamos National Laboratory, Los Alamos, NM 87545

^d   A. Tomkinson: University of Texas at San Antonio, San Antonio, TX 78285

^e   D. Beach: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998