The BLM dissolvasome in DNA replication and repair

RecQ DNA helicases are critical for proper maintenance of genomic stability, and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase IIIα, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems.     

Sister chromatid exchanges in human lymphocytes exposed to 8-methoxypsoralen and long wave UV radiation prior to incorporation of bromodeoxyuridine

Summary Human lymphocytes exposed to the effects of long wave UV radiation in the presence of 8-methoxypsoralen prior to stimulation by PHA show dose related sister chromatid exchanges after 2 replication cycles in vitro. This has implications for interpreting the repair processes involved and for monitoring DNA damaging agents in vivo.     

DNA repair nucleases

Stability of DNA largely depends on accuracy of repair mechanisms, which remove structural anomalies induced by exogenous and endogenous agents or introduced by DNA metabolism, such as replication. Most repair mechanisms include nucleolytic processing of DNA, where nucleases cleave a phosphodiester bond between a deoxyribose and a phosphate residue, thereby producing 5-terminal phosphate and 3-terminal hydroxyl groups. Exonucleases hydrolyse nucleotides from either the 5 or 3 end of DNA, while endonucleases incise internal sites of DNA. Flap endonucleases cleave DNA flap structures at or near the junction between single-stranded and double-stranded regions. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. In addition, nucleolytic repair functions are required during replication to remove misincorporated nucleotides, Okazaki fragments and 3 tails that may be formed after repair of stalled replication forks.Received 12 June 2003; received after revision 29 July 2003; accepted 16 September 2003     

Faithful after break-up: suppression of chromosomal translocations

Chromosome integrity in response to chemically or radiation-induced chromosome breaks and the perturbation of ongoing replication forks relies on multiple DNA repair mechanisms. However, repair of these lesions may lead to unwanted chromosome rearrangement if not properly executed or regulated. As these types of chromosomal alterations threaten the cell’s and the organism’s very own survival, multiple systems are developed to avoid or at least limit break-induced chromosomal rearrangements. In this review, we highlight cellular strategies for repressing DNA break-induced chromosomal translocations in multiple model systems including yeast, mouse, and human. These pathways select proper homologous templates or broken DNA ends for the faithful repair of DNA breaks to avoid undesirable chromosomal translocations.     

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.     

Welcome the Family of FANCJ-like Helicases to the Block of Genome Stability Maintenance Proteins

The FANCJ family of DNA helicases is emerging as an important group of proteins for the prevention of human disease, cancer, and chromosomal instability. FANCJ was identified by its association with breast cancer, and is implicated in Fanconi Anemia. Proteins with sequence similarity to FANCJ are important for maintenance of genomic stability. Mutations in genes encoding proteins related to FANCJ, designated ChlR1 in human and Chl1p in yeast, result in sister chromatid cohesion defects. Nematodes mutated in dog-1 show germline as well as somatic deletions in genes containing guanine-rich DNA. Rtel knockout mice are embryonic lethal, and embryonic stem cells show telomere loss and chromosomal instability. FANCJ also shares sequence similarity with human XPD and yeast RAD3 helicases required for nucleotide excision repair. The recently solved structure of XPD has provided new insight to the helicase core and accessory domains of sequence related Superfamily 2 helicases. The functions and roles of members of the FANCJ-like helicase family will be discussed. Received 17 September 2008; received after revision 24 October 2008; accepted 28 October 2008     

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals

The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.     

Targeting abnormal DNA double strand break repair in cancer

A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agents that selectively inhibit the DSB repair pathways that cancer cells are more dependent upon will facilitate the design of therapeutic strategies that exploit the differences in DSB repair between normal and cancer cells. Here, we discuss the pathways of DSB repair, alterations in DSB repair in cancer, inhibitors of DSB repair and future directions for cancer therapies that target DSB repair.     

<Emphasis Type="Italic">XPF</Emphasis> knockout via CRISPR/Cas9 reveals that ERCC1 is retained in the cytoplasm without its heterodimer partner XPF

The XPF/ERCC1 heterodimeric complex is essentially involved in nucleotide excision repair (NER), interstrand crosslink (ICL), and double-strand break repair. Defects in XPF lead to severe diseases like xeroderma pigmentosum (XP). Up until now, XP-F patient cells have been utilized for functional analyses. Due to the multiple roles of the XPF/ERCC1 complex, these patient cells retain at least one full-length allele and residual repair capabilities. Despite the essential function of the XPF/ERCC1 complex for the human organism, we successfully generated a viable immortalised human XPF knockout cell line with complete loss of XPF using the CRISPR/Cas9 technique in fetal lung fibroblasts (MRC5Vi cells). These cells showed a markedly increased sensitivity to UVC, cisplatin, and psoralen activated by UVA as well as reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the newly generated knockout cells, we could show that human XPF is markedly involved in homologous recombination repair (HRR) but dispensable for non-homologous end-joining (NHEJ). Notably, ERCC1 was not detectable in the nucleus of the XPF knockout cells indicating the necessity of a functional XPF/ERCC1 heterodimer to allow ERCC1 to enter the nucleus. Overexpression of wild-type XPF could reverse this effect as well as the repair deficiencies.     

Quality control of homologous recombination

Exogenous and endogenous genotoxic agents, such as ionizing radiation and numerous chemical agents, cause DNA double-strand breaks (DSBs), which are highly toxic and lead to genomic instability or tumorigenesis if not repaired accurately and efficiently. Cells have over evolutionary time developed certain repair mechanisms in response to DSBs to maintain genomic integrity. Major DSB repair mechanisms include non-homologous end joining and homologous recombination (HR). Using sister homologues as templates, HR is a high-fidelity repair pathway that can rejoin DSBs without introducing mutations. However, HR execution without appropriate guarding may lead to more severe gross genome rearrangements. Here we review current knowledge regarding the factors and mechanisms required for accomplishment of accurate HR.     

DNA damage repair and transcription

Double-strand breaks arise frequently in the course of endogenous - normal and pathological - cellular DNA metabolism or can result from exogenous agents such as ionizing radiation. It is generally accepted that these lesions represent one of the most severe types of DNA damage with respect to preservation of genomic integrity. Therefore, cells have evolved complex mechanisms that include cell-cycle arrest, activation of various genes, including those associated with DNA repair, and in certain cases induction of the apoptotic pathway to respond to double-strand breaks. In this review we discuss recent progress in our understanding of cellular responses to DNA double-strand breaks. In addition to an analysis of the current paradigms of detection, signaling and repair, insights into the significance of chromatin remodeling in the double-strand break-response pathways are provided.