Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. A. Introduction: Mammalian Topoisomerase Families, Top1 Functions and Catalytic MechanismsSeven topoisomerase genes are encoded in the human nuclear genome [1]. The enzymes (abbreviated Topo or Top) have been numbered in the order of their discovery except for the most recent enzyme, mitochondrial topoisomerase I (Top1mt) [2,3]. Vertebrate cells contain two Top1 (Top1 for the nuclear genome and Top1mt for the mitochondrial genome), two Top2 (Top2α and β) and two Top3 (Top3α and β). The seventh topoisomerase is Spo11, whose expression is restricted to germ cells. Top3α forms heterodimers with BLM (the gene product deficient in Bloom syndrome) and is functionally related to the resolution of post-replicative hemicatenanes and recombination intermediates [4,5]. Top1 proteins belong to the family of the tyrosine recombinases (which includes λ-integrase, Flip and Cre recombinases), and Top2 is related to bacterial gyrase and Topo IV, which are the targets of quinolone antibiotics.Topoisomerases and tyrosine recombinases nick and religate DNA by forming a covalent enzyme-DNA intermediate between an enzyme catalytic tyrosine residue and the end of the broken DNA (Fig. 1). These covalent intermediates are generally referred to as "cleavage (or cleavable) complexes" (Fig. 2). Topoisomerases have also been classified in two groups depending whether they cleave and religate one strand (type I) or both strands (type II) of the DNA duplex. Type I enzymes include Top1 (nuclear), Top1mt, Top3α and β and type II enzymes include Top2α and β and Spo11.Top1 is essential in vertebrates and flies but not in yeast. Knocking out the TOP1 gene results in early embryonic lethality in mouse [6] and fly [7]. By contrast, yeast survives in the absence *To whom reprint requests should be addressed, Bldg. 37, Rm. 5068, NIH, Bethesda, MD 20892-4255 [8]. Top1 is expressed constitutively throughout the cell cycle [9] and is concentrated in the nucleolus [10,11]. Its main function is to relieve both positive and negative DNA supercoiling generated by transcription and replication, and possibly DNA repair and chromatin remodeling [1,[12][13][14]. The mechanistic sim...

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4Ј-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiationinduced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.