A bifunctional antibody conjugate marks the location of DNA binding proteins on deproteinized DNA fibers.

Immunofluorescent foci of DNA Damage Response (DDR) proteins serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, Double Strand Breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX (γ-H2AX). The association with DSBs is so well established that the reverse interpretation that γ-H2AX invariably implies DSBs is routine.

Repair of genomic interstrand crosslinks.

Genomic interstrand crosslinks (ICLs) are formed by reactive species generated during normal cellular metabolism, produced by the microbiome, and employed in cancer chemotherapy. While there are multiple options for replication dependent and independent ICL repair, the crucial step for each is unhooking one DNA strand from the other. Much of our insight into mechanisms of unhooking comes from powerful model systems based on plasmids with defined ICLs introduced into cells or cell free extracts.

The Response of the Replication Apparatus to Leading Template Strand Blocks.

Duplication of the genome requires the replication apparatus to overcome a variety of impediments, including covalent DNA adducts, the most challenging of which is on the leading template strand. Replisomes consist of two functional units, a helicase to unwind DNA and polymerases to synthesize it. The helicase is a multi-protein complex that encircles the leading template strand and makes the first contact with a leading strand adduct.

Imaging the cellular response to an antigen tagged interstrand crosslinking agent.

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited.

An optimized proximity ligation assay to detect telomere dysfunction induced foci in human and mouse cells.

Telomere dysfunction-induced foci (TIF) can be measured by immunofluorescence, combined with telomere-fluorescent hybridization. We modified this approach by combining the proximity ligation assay (PLA), which detects colocalization of two molecules in proximity through a signal amplification step and improves the fidelity and sensitivity of TIF detection in human and mouse cells. The protocol includes cell preparation, permeabilization, fixation, and blocking PLA detection of DNA damage response proteins within proximity with telomeres and optional PLA verification by immunofluorescence-based technique.

RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse.

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress.

Visualizing replication fork encounters with DNA interstrand crosslinks.

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes.

RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair.

SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown.

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin.

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling-double strand breaks, reversed forks, or genomic rearrangements-there is limited understanding of the determinants of replisome stalling vs.

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites.

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored.

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes.

Defective postreplication repair of UV photoproducts in melanoma: a mutator phenotype.

In this issue, the Gabrielli laboratory and collaborators address the bulky CPD lesions created in DNA when UV joins two adjacent pyrimidines (thymine or cytosine), leading to skin cancers such as melanoma (Pavey S et al. (2019) Mol Oncol). Our understanding of postreplication repair mechanisms for bulky lesions has lagged, and the newly reported predominance of translational control in the UV response has important implications.

Replisome structure suggests mechanism for continuous fork progression and post-replication repair.

What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replication-fork progression, would stall at road blocks. After the discovery of replication-fork helicase and replication re-initiation factors by the 1990s, it became clear that the replisome can "skip" impasses and finish replication with single-stranded gaps and double-strand breaks in the product DNA.

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process.

Loss of ARID1A in Tumor Cells Renders Selective Vulnerability to Combined Ionizing Radiation and PARP Inhibitor Therapy.

Purpose: Somatic inactivating mutations in ARID1A, a component of the SWI/SNF chromatin remodeling complex, are detected in various types of human malignancies. Loss of ARID1A compromises DNA damage repair. The induced DNA damage burden may increase reliance on PARP-dependent DNA repair of cancer cells to maintain genome integrity and render susceptibility to PARP inhibitor therapy.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2.

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM.

Imaging cellular responses to antigen tagged DNA damage.

Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA.

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

Background: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined.

Mechanistic insights into how CMG helicase facilitates replication past DNA roadblocks.

Before leaving the house, it is a good idea to check for road closures that may affect the morning commute. Otherwise, one may encounter significant delays arriving at the destination. While this is commonly true, motorists may be able to consult a live interactive traffic map and pick an alternate route or detour to avoid being late.

Arsenite Binds to the RING Finger Domain of FANCL E3 Ubiquitin Ligase and Inhibits DNA Interstrand Crosslink Repair.

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents.

CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes.

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing.

Single Molecule Analysis of Laser Localized Psoralen Adducts.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells.

Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer.

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin.

Single Molecule Analysis of Laser Localized Interstrand Crosslinks.

DNA interstrand crosslinks (ICLs) block unwinding of the double helix, and have always been regarded as major challenges to replication and transcription. Compounds that form these lesions are very toxic and are frequently used in cancer chemotherapy. We have developed two strategies, both based on immunofluorescence (IF), for studying cellular responses to ICLs.