Ionizing Radiation Sensitivity of DNA Polymerase Lambda-Deficient Cells

Vermeulen, Christie; Bertocci, Barbara; Begg, Adrian C.; Vens, Conchita

doi:10.1667/rr1057r.1

Cited by 23 publications

(15 citation statements)

References 18 publications

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“…These structures provide novel insight into the structural characteristics of these analogs that determine their efficient incorporation into DNA as well as the structural basis of sugar selectivity in Pol . Given the roles of Pol in DNA base excision repair and non-homologous end joining repair (21,37), it is possible that the cellular sensitivity to AraC may partly depend on Pol . In particular, Pol may have a more profound impact on cellular sensitivity to AraC when used in combination with other DNA-damaging agents, such as mitoxantrone, which introduces DNA double strand breaks, and/or hydroxyurea or fludarabine, which affect cellular nucleotide pools.…”

Section: Resultsmentioning

confidence: 99%

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

García‐Díaz

Murray

Kunkel

et al. 2010

Journal of Biological Chemistry

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The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase (Pol ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol . Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol could modulate the cytotoxic effect of AraC.Nucleoside analogs are an important class of compounds that are clinically used for anticancer and antiviral treatments (1). For example, 3TC, 3 AZT, and D4T have been used for antiviral treatments (1). AraC ( Fig. 1) is used to treat leukemia (2), and dFdC ( Fig. 1) is used to treat various types of cancer, such as non-small cell lung cancer (3), breast cancer (4), and pancreatic cancer (5). The main mechanism of action of nucleoside analogs is to terminate the DNA elongation process after their incorporation into viral or cellular DNA (6 -8). For example, due to the lack of a 3Ј-hydroxyl group on their sugar moieties, the incorporation of the antiviral compounds 3TC, AZT, and D4T terminates human immunodeficiency virus DNA replication immediately (9).Although both AraC and gemcitabine have a 3Ј-hydroxyl group on their sugar moieties, the incorporation of either compound has been shown to terminate the DNA elongation processes and result in DNA fragmentation (7,10). Studies have shown that the arabinose sugar moiety in AraC and the difluoro group on the 2Ј-position of the sugar moiety of dFdC alter the DNA structure, inhibiting DNA polymerases (11,12). The cytotoxicity of AraC or dFdC is proportional to the amount of the analog incorporated into DNA (6 -8). Therefore, the cellular enzymes that are involved in activating these nucleoside analogs and the DNA polymerases that introduce them into DNA play a critical role in determining their cytotoxic activity.Human DNA polymerase (Pol ) is an exonuclease-deficient, 575-amino acid DNA polymerase that belongs to the X-family (13). It contains a N-terminal BRCT domain that is important for protein partnerships, a proline/serine-rich region that has been suggested to be a target for post-translational modification, and a C-terminal 39-kDa polymerase domain (14). The 39-kDa domain, which is structurally well characterized (15, 16) contains the fingers, palm, and thumb subdomains typical of DNA polymerases (17). It also contains an 8-kDa subdomain with dRP lyase activity (14) that contributes to base excision repair (BER) in vitro. Studies in Pol ␤-deficient mouse embryonic fibroblast extracts indicate that Pol also performs BER in vivo (18,19). Moreover, in a confocal microscopy study, Pol reloca...

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Section: Resultsmentioning

confidence: 99%

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

García‐Díaz

Murray

Kunkel

et al. 2010

Journal of Biological Chemistry

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“…We conclude from these data that Pol ␤ can function independently of interaction with XRCC1 following oxidative DNA damage or, more likely, that another DNA polymerase can substitute for Pol ␤. For example, Pol , Pol , and Pol Q also are implicated in the cellular response to H 2 O 2 , and Pol Q has been reported to physically associate with XRCC1 (5,39,47,51,57). Alternatively, the long-patch repair DNA polymerases Pol ␦ and/or Pol ε might substitute for Pol ␤.…”

Section: Discussionmentioning

confidence: 99%

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Breslin

Caldecott

2009

Molecular and Cellular Biology

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Oxidative stress is a major source of chromosome single-strand breaks (SSBs), and the repair of these lesions is retarded in neurodegenerative disease. The rate of the repair of oxidative SSBs is accelerated by XRCC1, a scaffold protein that is essential for embryonic viability and that interacts with multiple DNA repair proteins. However, the relative importance of the interactions mediated by XRCC1 during oxidative stress in vivo is unknown. We show that mutations that disrupt the XRCC1 interaction with DNA polymerase ␤ or DNA ligase III fail to slow SSB repair in proliferating CHO cells following oxidative stress. In contrast, mutation of the domain that interacts with polynucleotide kinase/phosphatase (PNK) and Aprataxin retards repair, and truncated XRCC1 encoding this domain fully supports this process. Importantly, the impact of mutating the protein domain in XRCC1 that binds these end-processing factors is circumvented by the overexpression of wild-type PNK but not by the overexpression of PNK harboring a mutated DNA 3-phosphatase domain. These data suggest that DNA 3-phosphatase activity is critical for rapid rates of chromosomal SSB repair following oxidative stress, and that the XRCC1-PNK interaction ensures that this activity is not rate limiting in vivo.Oxidative stress can have a major influence on genome integrity and cell survival and is an etiological factor in a number of neurological human diseases. Of these, several are associated with defects in the repair of DNA damage, including xeroderma pigmentosum (XP), ataxia telangiecatsia (A-T), ataxia oculomotor apraxia 1 (AOA1), and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) (1,28,34,37). The neuropathology evident in XP most likely reflects an inability to repair one or more single-strand oxidative adducts by nucleotide excision repair. In contrast, A-T is associated with cellular defects in the repair of DNA double-strand breaks (DSBs) and AOA1 and SCAN1 with defects in the repair of DNA single-strand breaks (SSBs). SSBs are the commonest DNA lesions arising in cells, and if they are not rapidly repaired they can inhibit transcription and/or generate replication-associated DSBs (3,26,59,60). The repair of oxidative SSBs involves DNA damage detection by PARP-1 followed by recruitment of the enzymes required for subsequent steps of the repair process, which include DNA end processing, DNA gap filling, and DNA ligation (9, 19). Many of the enzymes implicated in these steps interact physically with XRCC1, including DNA polynucleotide kinase (PNK) (54), Aprataxin (APTX) (13,14,18,31,44), DNA polymerase ␤ (Pol ␤) (10, 27), and DNA ligase III␣ (Lig3␣) (11,12). This has prompted the hypothesis that XRCC1 is a scaffold protein that recruits, stabilizes, and/or stimulates SSB repair (SSBR) enzymes at chromosomal SSBs, thereby accelerating the overall process (8, 9). While in vitro analyses generally are consistent with this idea, including the observation that XRCC1 mutation (50, 58), deletion (49), or depletion (6) retards the rate of chromos...

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“…Third, in an in vitro system in which the joining of incompatible ends is dependent on Ku, Artemis, DNA-Pkcs, XRCC4, and DNA ligase IV, addition of Pol λ preserves the 5′ overhang and increases the size of the junctions [152]. Fourth, Pol λ deficient mouse embryo fibroblasts are sensitive to hydrogen peroxide, camptothecin, and etoposide, but they are not sensitive to ionizing radiation (IR) [154, 155]. All of these agents produce breaks in DNA, so the sensitivity of Pol λ-deficient cells suggests that it functions during the repair of double- and single-strand breaks.…”

Section: Dna Polymerase λmentioning

confidence: 99%

DNA polymerase Family X: Function, structure, and cellular roles

Yamtich

Sweasy

2010

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

133

151

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The X Family of DNA polymerases in eukaryotic cells consists of Terminal Transferase, and DNA polymerases β, λ, and μ. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases β, λ, and μ. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.

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Ionizing Radiation Sensitivity of DNA Polymerase Lambda-Deficient Cells

Cited by 23 publications

References 18 publications

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

DNA polymerase Family X: Function, structure, and cellular roles

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