Essay Example on For DNA replication to be accurately Completed

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Requires the action of many molecular players that need to exert their functions in a perfectly concerted manner For DNA replication to be accurately completed the replication fork must frequently overcome a multitude of structurally unrelated obstacles such as DNA lesions transcribing RNA polymerases and tightly bound protein DNA complexes Stalling of the replication machinery during S phase creates a hazardous situation for cells initiating a subsequent replication fork collapse resulting in genomic instability such as copy number variation Arlt 2011 1 micronuclei formation Xu 2011 2 and loss of heterozygosity Donahue 2006 3 thus contributing to tumorigenesis Kawabata 2011 4 Camptothecin CPT and its derivatives that have been used in anticancer therapy for more than fifteen years are highly selective inhibitors of topoisomerase I TopI that lock the enzyme on DNA during the intermediate step of enzymatic cleavage 



This leads to formation of irreversible TopI DNA cleavable complexes These bulky DNA lesions block vital cellular processes notably DNA replication and transcription reviewed in Tomicic 2013 5 In proliferating cells a collision of replication machinery with these complexes is a primary reason for formation of double strand breaks In contrast to the double strand breaks produced by ionizing radiation or Sce I digestion these are one ended making them a poor substrate for non homologous end joining NHEJ Such lesions are therefore repaired primarily by HR The outcome of CPT therapy is known to depend on p53 status with p53 or p21Waf1 mutant cells being more sensitive to therapy than p53 proficient ones The differential sensitivity to CTP therapy is attributed to the ability of p53 proficient cells to undergo an extended G2 arrest thus providing cells additional time for DNA repair Human RPA consisting of 70 kDa RPA1 30 kDa RPA2 and 14 kDa RPA3 subunits is subject to extensive phosphorylation on its middle subunit RPA2 Binz 2004 6 The N terminal RPA2 domain undergoes both cell cycle and stress dependent phosphorylation Bochkareva 2002 7 Bochkareva 2005 8 Ser23 and Ser29 are constitutively modified during mitosis by cyclin B CDK1 Fang 1993 9 Oakley 2003 10 Murphy 2014 58 and at the entrance to S phase by the cyclin A CDK2 complex Fang 1993 9 Dutta 1992 11 Niu 1997 12 These two residues also undergo phosphorylation in response to genotoxic stresses such as UV irradiation bleomycin treatment or replication arrest induced by CPT Abramova 1997 13 Anantha 2007 14 The primary activation pathway during DNA replication stress apparently involves binding of RPA to ssDNA formed as a result of DNA polymerase stalling and continued movement of the replicative helicase 



These persistent RPA ssDNA intermediates recruit the ATR ATRIP complex leading to activation of the ATR kinase Zou 2003 15 which phosphorylates RPA2 at Ser33 and activates CHK1 an S phase checkpoint regulator Olson 2006 16 Zernik Kobak 1997 17 A collapse of replication forks and the resulting formation of double strand breaks stimulates ATM and DNA PKcs kinases that induce CHK2 another S phase checkpoint regulator Under DNA damage conditions the phosphorylation of Thr21 is apparently catalyzed by ATM DNA PKcs and probably ATR Zernik Kobak 1997 17 Block 2004 18 The remaining sites Ser4 Ser8 Ser11 Ser12 and Ser13 are phosphorylated in response to genotoxic stress presumably by DNA PKcs An investigation of the functional role of RPA phosphorylation in response to replication arrest has demonstrated its critical role in DNA repair by HR as well as in a consequent restoration of DNA synthesis and cell viability Vassin 2009 50 Anantha 2007 14 P53 is well recognized as a protein with pleiotropic functions in DNA repair replication and recombination Albrechtsen 1999 20 Dudenhoffer 1998 21 Liu 2000 22

Data from our own and other laboratories demonstrated that p53 suppresses spontaneous inter and intra molecular HR indicating this may be one mechanism by which p53 maintains genomic integrity Bertrand 1997 23 Dudenhoffer 1998 21 Mekeel 1997 25 The mechanisms by which p53 regulates HR are just beginning to be elucidated An assessment of p53 involvement in HR under conditions of genotoxic stress has always been complicated by its other roles such as regulation of the cell cycle and apoptosis Probably due to the deficiency in DNA double strand end resection in the G1 stage of cell cycle DNA repair by HR is restricted to S and G2 stages Therefore under genotoxic stress cell accumulation in G1 was suggested to account for the inhibitory effect of p53 on HR that was observed in many experiments Rieckmann 2013 26 However a number of findings suggest that p53 protein may be directly involved in recombination control It has been reported that p53 binds to recombination intermediates and Holliday junctions in vitro that is required for efficient inhibition of HR in vivo Lee 1997 27 Dudenhoffer 1998 21 Susse 2000 28 Hampp 2016 29 In addition p53 interacts with various proteins involved in HR such as the Rad51 recombinase the helicases mutated in Werner and Bloom syndromes and RPA Dutta 1993 30 Leiter 1996 31 Sturzbecher 1996 32 Brush 1994 33


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