scholarly journals APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

2008 ◽  
Vol 28 (14) ◽  
pp. 4620-4628 ◽  
Author(s):  
Stuart L. Rulten ◽  
Felipe Cortes-Ledesma ◽  
Liandi Guo ◽  
Natasha J. Iles ◽  
Keith W. Caldecott
Keyword(s):  
Zinc Finger ◽  
Mammalian Cells ◽  
Strand Break ◽  
Zinc Finger Domain ◽  
Chromosomal Damage ◽  
Chromosomal Dna ◽  
Fha Domain ◽  
Break Repair ◽  
Dna Strand ◽  
Tandem Zinc Finger

ABSTRACT APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.

scholarly journals APLF (C2orf13) Is a Novel Human Protein Involved in the Cellular Response to Chromosomal DNA Strand Breaks

2007 ◽  
Vol 27 (10) ◽  
pp. 3793-3803 ◽  
Author(s):  
Natasha Iles ◽  
Stuart Rulten ◽  
Sherif F. El-Khamisy ◽  
Keith W. Caldecott
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Strand Break ◽  
Dna Strand Breaks ◽  
Double Strand Break Repair ◽  
Chromosomal Dna ◽  
Fha Domain ◽  
Strand Breaks ◽  
Break Repair ◽  
Dna Strand

ABSTRACT Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H2O2 or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.

scholarly journals Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

2001 ◽  
Vol 21 (10) ◽  
pp. 3425-3435 ◽  
Author(s):  
Mark D. Baker ◽  
Erin C. Birmingham
Keyword(s):  
Homologous Recombination ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Holliday Junction ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Break Repair

ABSTRACT In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

scholarly journals Use of a Small Palindrome Genetic Marker to Investigate Mechanisms of Double-Strand-Break Repair in Mammalian Cells

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1281-1289 ◽  
Author(s):  
Julang Li ◽  
Mark D Baker
Keyword(s):  
Mismatch Repair ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Chromosomal Markers ◽  
Vector Borne ◽  
Break Repair ◽  
The One

Abstract We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.

scholarly journals Testing Predictions of the Double-Strand Break Repair Model Relating to Crossing Over in Mammalian Cells

Genetics ◽  
2004 ◽  
Vol 168 (3) ◽  
pp. 1539-1555 ◽  
Author(s):  
Erin C. Birmingham ◽  
Shauna A. Lee ◽  
Richard D. McCulloch ◽  
Mark D. Baker
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Crossing Over ◽  
Break Repair

scholarly journals Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair

2020 ◽  
Vol 48 (12) ◽  
pp. 6672-6684 ◽  
Author(s):  
Ilona Kalasova ◽  
Richard Hailstone ◽  
Janin Bublitz ◽  
Jovel Bogantes ◽  
Winfried Hofmann ◽  
...  
Keyword(s):  
Strand Break ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Fha Domain ◽  
Single Strand Break ◽  
Strand Breaks ◽  
Charcot Marie Tooth Disease ◽  
Single Strand Break Repair ◽  
Primary Determinant ◽  
Break Repair

Abstract Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.

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