PARP/XRCC1 surveillance of the human genome

DNA single strand breaks (SSBs) are one of the most common lesions to genomic DNA, arising from various endogenous and exogenous sources. Single strand break repair (SSBR) constitutes a biochemical pathway whereby SSBs are detected, enzymatically processed and ligated. Whilst the general mechanisms of SSBR are relatively well described in vitro, there are remaining questions concerning how the pathway operates in vivo. For example, an early step in SSBR is thought to be the poly(ADP-ribose) polymerase (PARP)-dependent modification of SSB-proximal proteins with ADP-ribose, which is a signal for the recruitment of downstream repair factors, including the central scaffold XRCC1. Yet, the presence of multiple DNA-dependent PARP genes (PARP1, PARP2 and PARP3) has caused confusion regarding their specific roles in SSBR. This thesis potentially clarifies some contentious aspects of PARP function in the repair of SSBs induced by reactive oxygen species (ROS) and by Topoisomerase 1 (Top1). By employing PARP1-/-/PARP2-/- cells generated herein using CRISPR-Cas9 technology, in combination with preextraction immunofluorescence imaging and high-content analysis, I demonstrate that both PARP1 and PARP2 contribute towards ROS-induced ADP-ribosylation and XRCC1 chromatin-localization, but that in response to Top1-SSBs, these functions are specifically supported by PARP1 alone. Furthermore, using TDP1-/- and XRCC1-/-/TDP1-/- cells also generated herein, I characterize a striking hyper-ADP-ribosylation phenotype in response to Top1-SSBs. The clinical significance of this was confirmed by co-workers, who observed a similar phenotype in an XRCC1-deficient patient, where mutations in XRCC1 underlie a novel cerebellar neurodegenerative disease. This phenotype could be utilized in future to screen for genes with novel functions in SSBR. Finally, I investigate the functional implications of disrupted SSBR genes for rates of repair and cellular viability using alkaline single-cell electrophoresis and clonogenic survival assay. In doing so, I unexpectedly discovered that deletion of PARP1 suppresses CPT-induced comet tail moments of WT and XRCC1-/- cells

A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo

Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin

A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand breakinduced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established ‘isogenic’ human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably,concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress

Exact Gap Computation for Code Coverage Metrics in ISO-C

Test generation and test data selection are difficult tasks for model based testing. Tests for a program can be meld to a test suite. A lot of research is done to quantify the quality and improve a test suite. Code coverage metrics estimate the quality of a test suite. This quality is fine, if the code coverage value is high or 100%. Unfortunately it might be impossible to achieve 100% code coverage because of dead code for example. There is a gap between the feasible and theoretical maximal possible code coverage value. Our review of the research indicates, none of current research is concerned with exact gap computation. This paper presents a framework to compute such gaps exactly in an ISO-C compatible semantic and similar languages. We describe an efficient approximation of the gap in all the other cases. Thus, a tester can decide if more tests might be able or necessary to achieve better coverage.Comment: In Proceedings MBT 2012, arXiv:1202.582

HIV-1 Epidemic in the Caribbean Is Dominated by Subtype B

The molecular epidemiology of HIV-1 in the Caribbean has been described using partial genome sequencing; subtype B is the most common subtype in multiple countries. To expand our knowledge of this, nearly full genome amplification, sequencing and analysis was conducted.Virion RNA from sera collected in Haiti, Dominican Republic, Jamaica and Trinidad and Tobago were reverse transcribed, PCR amplified, sequenced and phylogenetically analyzed. Nearly full genomes were completed for 15 strains; partial pol was done for 67 strains. All but one of the 67 strains analyzed in pol were subtype B; the exception was a unique recombinant of subtypes B and C collected in the Dominican Republic. Of the nearly full genomes of 14 strains that were subtype B in pol, all were subtype B from one end of the genome to the other and not inter-subtype recombinants. Surprisingly, the Caribbean subtype B strains clustered significantly with each other and separate from subtype B from other parts of the pandemic.The more complete analysis of HIV-1 from 4 Caribbean countries confirms previous research using partial genome analysis that the predominant subtype in circulation was subtype B. The Caribbean strains are phylogenetically distinct from other subtype B strains although the biological meaning of this finding is unclear

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair1,2. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP3,4,5 and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease

Altered microbiomes in thirdhand smoke-exposed children and their home environments.

IntroductionTobacco smoke contains numerous toxic chemicals that accumulate in indoor environments creating thirdhand smoke (THS). We investigated if THS-polluted homes differed in children's human and built-environment microbiomes as compared to THS-free homes.MethodsParticipants were n = 19 THS-exposed children and n = 10 unexposed children (≤5 years) and their caregivers. Environmental and biological samples were analyzed for THS pollutants and exposure. Swab samples were collected from the built-environment (floor, table, armrest, bed frame) and child (finger, nose, mouth, and ear canal), and 16S ribosomal RNA genes were analyzed for bacterial taxa using high-throughput DNA sequencing.ResultsPhylogenetic α-diversity was significantly higher for the built-environment microbiomes in THS-polluted homes compared to THS-free homes (p < 0.014). Log2-fold comparison found differences between THS-polluted and THS-free homes for specific genera in samples from the built-environment (e.g., Acinetobacter, Bradyrhizobium, Corynebacterium, Gemella, Neisseria, Staphylococcus, Streptococcus, and Veillonella) and in samples from children (esp. Corynebacterium, Gemella, Lautropia, Neisseria, Rothia, Staphylococcus, and Veillonella).ConclusionWhen exposed to THS, indoor and children microbiomes are altered in an environment-specific manner. Changes are similar to those reported in previous studies for smokers and secondhand smoke-exposed persons. THS-induced changes in child and built-environmental microbiomes may play a role in clinical outcomes in children.ImpactDespite smoking bans, children can be exposed to tobacco smoke residue (i.e., thirdhand smoke) that lingers on surfaces and in settled house dust. Thirdhand smoke exposure is associated with changes in the microbiomes of the home environment and of the children living in these homes. Thirdhand smoke is associated with increased phylogenetic diversity of the home environment and changes in the abundances of several genera of the child microbiome known to be affected by active smoking and secondhand smoke (e.g., Corynebacterium, Staphylococcus, Streptococcus). Thirdhand smoke exposure by itself may induce alterations in the microbiome that play a role in childhood pathologies

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like nucleoid-associated protein that likely influences phage excision-integration reactions or bacterial gene expression