Beyond base excision repair: an evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.     

Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers

In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase eta, were devoid of bypass activity. Bypass-proficient extracts as a group (n=16 for 3 extracts) displayed higher efficiency (P=0.005) for replication past the [c,s]TT during leading strand synthesis (84+/-22%) than during lagging strand synthesis (64+/-13%). These findings are compared to previous results concerning the bypass of the (6-4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.     

Histone deacetylases 1 and 2 regulate DNA replication and DNA repair: potential targets for genome stability-mechanism-based therapeutics for a subset of cancers

Histone deacetylases 1 and 2 (HDAC1,2) belong to the class I HDAC family, which are targeted by the FDA-approved small molecule HDAC inhibitors currently used in cancer therapy. HDAC1,2 are recruited to DNA break sites during DNA repair and to chromatin around forks during DNA replication. Cancer cells use DNA repair and DNA replication as survival mechanisms and to evade chemotherapy-induced cytotoxicity. Hence, it is vital to understand how HDAC1,2 function during the genome maintenance processes (DNA replication and DNA repair) in order to gain insights into the mode-of-action of HDAC inhibitors in cancer therapeutics. The first-in-class HDAC1,2-selective inhibitors and Hdac1,2 conditional knockout systems greatly facilitated dissecting the precise mechanisms by which HDAC1,2 control genome stability in normal and cancer cells. In this perspective, I summarize the findings on the mechanistic functions of class I HDACs, specifically, HDAC1,2 in genome maintenance, unanswered questions for future investigations and views on how this knowledge could be harnessed for better-targeted cancer therapeutics for a subset of cancers.     

Coordinating DNA polymerase traffic during high and low fidelity synthesis

With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell's genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS.     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.     

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD*

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.     

The checkpoint response to replication stress

Genome instability is a hallmark of cancer cells, and defective DNA replication, repair and recombination have been linked to its etiology. Increasing evidence suggests that proteins influencing S-phase processes such as replication fork movement and stability, repair events and replication completion, have significant roles in maintaining genome stability. DNA damage and replication stress activate a signal transduction cascade, often referred to as the checkpoint response. A central goal of the replication checkpoint is to maintain the integrity of the replication forks while facilitating replication completion and DNA repair and coordinating these events with cell cycle transitions. Progression through the cell cycle in spite of defective or incomplete DNA synthesis or unrepaired DNA lesions may result in broken chromosomes, genome aberrations, and an accumulation of mutations. In this review we discuss the multiple roles of the replication checkpoint during replication and in response to replication stress, as well as the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.     

DNA bending facilitates the error‐free DNA damage tolerance pathway and upholds genome integrity

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.     

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.     

Coordination of DNA replication and recombination activities in the maintenance of genome stability

Across the evolutionary spectrum, living organisms depend on high-fidelity DNA replication and recombination mechanisms to maintain genome stability and thus to avoid mutation and disease. The repair of severe lesions in the DNA such as double-strand breaks or stalled replication forks requires the coordinated activities of both the homologous recombination (HR) and DNA replication machineries. Growing evidence indicates that so-called "accessory proteins" in both systems are essential for the effective coupling of recombination to replication which is necessary to restore genome integrity following severe DNA damage. In this article we review the major processes of homology-directed DNA repair (HDR), including the double Holliday Junction (dHJ), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and error-free lesion bypass pathways. Each of these pathways involves the coupling of a HR event to DNA synthesis. We highlight two major classes of accessory proteins in recombination and replication that facilitate HDR: Recombination mediator proteins exemplified by T4 UvsY, Saccharomyces cerevisiae Rad52, and human BRCA2; and DNA helicases/translocases exemplified by T4 Gp41/Gp59, E. coli DnaB and PriA, and eukaryotic Mcm2-7, Rad54, and Mph1. We illustrate how these factors help to direct the flow of DNA and protein-DNA intermediates on the pathway from a double-strand break or stalled replication fork to a high-fidelity recombination-dependent replication apparatus that can accurately repair the damage.     

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein–protein and protein–DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis‐acting sequences that serve as replication origins and the trans‐acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed. J. Cell. Biochem. 106: 512–520, 2009. © 2009 Wiley‐Liss, Inc.     

Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease–mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination–deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.     

Mechanisms of Dealing with DNA Damage-Induced Replication Problems

During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.     

Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism

This review deals with analysis of mechanisms involved in coordination of DNA replication and repair by SSB proteins; characteristics of eukaryotic, prokaryotic, and archaeal SSB proteins are considered, which made it possible to distinguish general mechanisms specific for functioning of proteins from organisms of different life domains. Mechanisms of SSB protein interactions with DNA during metabolism of the latter are studied; structural organization of the SSB protein complexes with DNA, as well as structural and functional peculiarities of different SSB proteins are analyzed.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

Essential functions of iron-requiring proteins in DNA replication,repair and cell cycle control

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these ironrequiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.     

Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

Building up and breaking down: mechanisms controlling recombination during replication

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.     

组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。