RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.
Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G 2 arrest in HCT116 human colon cancer cells, as well as Mlh1 ؊/؊ murine embryonic fibroblasts, after treatment with 5-fluoro-2-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2 In addition to its roles in correcting DNA replication errors and editing recombination intermediates, DNA mismatch repair (MMR) 1 can process numerous DNA lesions (1-4). In fact, an intact MMR system is required for the lethality of specific DNA-damaging agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), 6-thioguanine (6-TG), and cisplatin (5-7). MMR also mediates the lethality of fluoropyrimidines (FPs) such as 5-fluorouracil (FU) and 5-fluoro-2Ј-deoxyuridine (FdUrd) (8, 9). Inactivation of MMR allows resistance to the cytotoxic effects of these agents, a phenomenon referred to as "damage tolerance" (10 -13). Importantly, this enables cancer cells to uncouple persistent DNA damage from cell death, resulting in increased drug resistance (14 -16).The two major gene products that comprise MMR are MSH2 (which heterodimerizes with MSH3 or MSH6 to recognize mispairs and loops in DNA) and MLH1 (which heterodimerizes with PMS2 or MLH3 to act as a molecular matchmaker between the MSH2 complex and other DNA repair/replication and perhaps cell cycle factors) (17, 18). Defects in these two genes account for most cases of hereditary non-polyposis colorectal cancer, a familial condition with a predisposition to cancers of the colon, endometrium, stomach, ovary, and biliary tracts (19), as well as sporadic tumors of the colon (20), endometrium (21), stomach (22), head and neck (23), and prostate (24).Others and we (8, 9) have demonstrated that cells deficient in MLH1 are resistant to the cytotoxic effects of FU and FdUrd. Because FPs are the agents of choice in the treatment of colorectal cancer, understanding potential resistance mechanisms is important. FPs exert cytotoxic effects through incorporation into RNA and/or DNA, as well as inhibition of thymidylate synthase (TS). The inhibition of TS, which is the central enzyme of de novo pyrimidine synthesis, leads to decreases in intracellular dTTP pools; this depletion results in immediate cytostatic effects (via inhibition of DNA synthesis) and alters dNTP pool sizes (thus increasing the error rate of DNA polymerase) (25). A hallmark of MMR deficiency is instability in the length of repetitive sequences in DNA, referred to as microsatellite instability (MSI). This reflects the inability of MMRdeficient cells to correct insertions and deletions in their DNA that result from polymerase slippage at these sequences (26). It is also an easily measured clinical marker. Due to the resistance of MMR-deficient (i.e. MSI ϩ ) cancer cells to FU and FdUrd, one woul...
Exonucleolytic degradation of DNA is an essential part of many DNA metabolic processes including DNA mismatch repair (MMR) and recombination. Human exonuclease I (hExoI) is a member of a family of conserved 5 3 3 exonucleases, which are implicated in these processes by genetic studies. Here, we demonstrate that hExoI binds strongly to hMLH1, and we describe interaction regions between hExoI and the MMR proteins hMSH2, hMSH3, and hMLH1. In addition, hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo. The study of interaction regions suggests a biochemical mechanism of the involvement of hExoI as a downstream effector in MMR and/or DNA recombination.DNA is susceptible to exogenous and endogenous damaging agents as well as spontaneous hydrolysis resulting in nucleotide damage, abasic (AP) sites, and DNA strand breaks (1). In addition, DNA sequence alterations may be caused by nucleotide misincorporation errors during DNA replication (2) and recombination between divergent DNA parents (3). Highly conserved DNA repair mechanisms have been identified, and inactivation of these pathways has been linked to a variety of diseases including cancer (4). DNA mismatch repair (MMR) 1 is one of the best studied repair pathways (5-7), and inactivating mutations in the human MMR genes are causally involved in the development of sporadic and hereditary cancers such as the common cancer susceptibility syndrome hereditary nonpolyposis colon cancer (HNPCC) (8).The biochemistry of MMR has been most extensively studied in Escherichia coli. Eight protein fractions (MutS, MutL, MutH, DNA helicase II (UvrD), single-strand DNA-binding protein, exonuclease I, DNA ligase, and the DNA polymerase III holoenzyme complex), ATP, and the four deoxynucleotide triphosphates are sufficient to carry out the complete MMR process in vitro (9). Several human MutS homologs (MSH) have been identified including the nuclear MMR proteins hMSH2, hMSH3, and hMSH6, as well as the meiosis-specific proteins hMSH4 and hMSH5. In eukaryotes, the MutS homologs function as heterodimers, which recognize single base mismatches, insertion/deletion loop-type mismatches, and some types of nucleotide damage (5, 10). The mechanism for the initial step(s) of MMR continues to be controversial (6, 11). However, there has been significant progress in deciphering the molecular basis for mispair recognition (12, 13) and the role of ATP binding and hydrolysis by the MutS homologs (14 -18). In contrast, the steps following the recognition of a mismatch or nucleotide lesion remain unclear. MutL homologs appear to be involved in signal transfer to downstream effectors (19 -23). In E. coli, MutL stimulates the endonucleolytic activity of MutH at GATC sites (24, 25). The resulting strand break functions as a signal for UvrD and exonucleases to remove the nascent strand starting at the GATC site and ending several nucleotides past the mismatch (26, 27). Four single-strand DNA exonucleases appear to maintain overlapping and redundant requirements for MMR: ExoI, RecJ, ExoV...
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