Incision Coordination in Nucleotide Excision Repair

This thesis aims to contribute to the understanding of the molecular mechanism that underlies one of the main DNA repair pathways in mammals, nucleotide excision rcpair. In chapter 1 the relevance of DNA repair in general is outlined. An overview of mammalian strategies to counteract DNA damage is provided, to show that an intricate network of repair machineries permanently guards the integrity of the genome. In discussing each repair pathway, attention is focussed on how DNA damage is removed and what protein fhetors arc required to accomplish this. Chapter I serves as a framework for chapter 2, in which one repair pathway, mammalian nucleotide excision repair, is discussed more extensively. In this chapter, a comprehensive oven,jew of the characteristics of each protein factor involve

Епічні аспекти сучасної української драми (на прикладі п’єси Анатолія Крима «Нелегалка»)

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DNA structural elements required for ERCC1-XPF endonuclease activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning o

Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients

Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease

Small chromosomal regions position themselves autonomously according to their chromatin class

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) genedepleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes

Annexin A5 haplotypes in the antiphospholipid syndrome

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Mapping of interaction domains between human repair proteins ERCC1 and XPF

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-b