Reparacion del dna

IUBMB

Life, 60(2): 130–134, February 2008

Research Communication
Trapping of Human DNA Topoisomerase I by DNA Structures Mimicking Intermediates of DNA Repair
Natalia Lebedeva1,2, Nadejda Rechkunova1, Serge Boiteux3 and Olga Lavrik1
1 2

Institute of Chemical Biology and Fundamental Medicine, Prospect Lavrentieva 8, 630090, Novosibirsk, Russia Department of Molecular Biology,Novosibirsk State University, 630090, Novosibirsk, Russia 3 ´culaire, BP6, 92265-Fontenay aux Roses, France CEA, UMR217 CNRS Institut de Radiobiologie Cellulaire et Mole

Summary In this report we show that human DNA Topoisomerase I (Top1) forms DNA-protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products wereidentified by SDS-gel electrophoresis. The chemical structure of the groups at 50 or 30 end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and othernick-sensors. Top1-DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability. Ó 2008 IUBMB IUBMB Life, 60(2): 130–134, 2008 Keywords
DNA topoisomerase 1; DNA repair; nicked DNA; DNA replication; enzymology.

INTRODUCTION DNA topoisomerases (including Top1) are ubiquitous enzymes involved in multiple processes, such as DNA replication, transcription, andrecombination (1–3). Top1 removes superhelical tension in chromosomal DNA during replication and transcription. The enzyme catalyzes this reaction by forming a transient reaction intermediate in which the 30 -phosphoryl group of the broken DNA strand is covalently linked to a tyrosine of the Top1 active site through a tyrosyl phosphate linkage. These intermediates are commonly referred to as Top1-DNAcleavage complexes (1–3). Under physiological conditions, Top1
Received 31 July 2007; accepted 19 October 2007 Address correspondence to: Olga Lavrik; Institute of Chemical Biology and Fundamental Medicine, 8, Lavrentiev Av., Novosibirsk, 630090, Russian Federation, Russia. Tel: 17-383-3309296. Fax: 17-383-3333677. E-mail: lavrik@niboch.nsc.ru
ISSN 1521-6543 print/ISSN 1521-6551 online DOI:10.1002/iub.5

catalyzes the religation of the cleaved DNA strand. However, a fraction of Top1-DNA cleavage complexes may also be converted into irreversible DNA damage during replication and transcription (4–6). The anticancer drug camptothecin (CPT) reversibly stabilizes the transient Top1-DNA cleavage complex (7). In addition to CPT, many DNA adducts, DNA damage or aberrant DNA structures such asuracil, 8-oxoguanine, mismatched bases or nicks also stabilize Top1-DNA cleavable complexes (8–11). These Top1DNA irreversible complexes if not rapidly degraded may promote illegitimate recombination and ultimately cause cell death, so become a cellular poison (12–14). We recently reported that yeast Top1 from cell free extracts of Saccharomyces cerevisiae can be trapped by nick-containing DNAs,but not by DNAs containing uracil, an AP site or a U.G mismatch (15). Our data suggest an interaction of yeast Top1 with DNA structures which appear in the course of the base excision repair (BER) process (15). This result is clearly different from previous studies showing that mammalian Top1 can be efficiently trapped on DNA that contains uracil, AP sites or nicks inserted in the vicinity of acanonical Top1 cleavable site (8–10). According to current models, BER intermediates are processed and then sequentially handed from one protein to the next in base excision repair pathways (16). Therefore, coordination of base excision repair proteins around BER DNA intermediates in the pathway is critical for preventing the exposure of nicked and one-nucleotide gapped DNAs to proteins such as...