Endogenous DNA Damage and Repair Enzymes—A short summary of the scientific achievements of Tomas Lindahl,Nobel Laureate in Chemistry 2015

Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career.     

That's A Scientist Should Do——A Dialog with Tomas Lindahl

正Dr Tomas Lindahl is a world-renowned scientist specialized in cancer research,in particular,DNA repair [1].In 2015,he was awarded the Nobel Prize in Chemistry jointly with Dr Paul L.Modrich and Dr Aziz Sancar ‘‘for mechanistic studies of DNA repair"(https://www.nobelprize.org/prizes/chemistry/2015/press-release/)[2].During his recent visits in China,besides delivering lectures,Tomas also attended a couple of group meetings with students and PIs.He actively interacted with the audience,not only on topics relating to DNA repair,but also on how to do science and beyond.We presented this special report based on recordings.     

The Nobel Prize in Chemistry 2015: Exciting discoveries in DNA repair by Aziz Sancar

正INTRODUCTION On October 7,2015,the Nobel Prize in Chemistry for 2015was awarded to three deserving scientists for their pioneering research on DNA repair.Tomas Lindahl was recognized for studies that uncovered the inherent instability of DNA as well as the mechanism of the base excision repair pathway,Paul Modrich for characterization of the mismatch repair     

Celebrating the work of Nobel Laureate Paul Modrich

正On October 7,2015,the Nobel Prize Committee announced that the 2015 Nobel Prize in Chemistry had been awarded jointly to Professors Paul Modrich,Tomas Lindahl and Aziz Sancar(Figure 1),each of whom made ground-breaking discoveries about the molecular mechanisms of DNA Repair.In one of life’s unpredictable turns,Paul was among the last individuals in his entire personal,professional and     

Structure and mechanism for DNA lesion recognition

A fundamental question in DNA repair is how a lesion is detected when embedded in millions to billions of normal base pairs. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This review summarizes seemingly diverse structural motifs used in lesion recognition and suggests a general mechanism to recognize DNA lesion by the poor base stacking. After initial recognition of this shared structural feature of lesions, different DNA repair pathways use unique verification mechanisms to ensure correct lesion identification and removal.     

Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-homologous DNA end joining： relevance to cancer, aging, and the immune system

Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

My Journey to DNA Repair

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases Ⅰ, and Ⅳ. I discovered the mammalian exonucleases DNase Ⅲ (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine (O6 mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.     

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.     

三名学者获诺贝尔化学奖 表彰其DNA修复研究

<正>北京时间10月7日下午,瑞典皇家科学院将今年的"诺贝尔化学奖"颁发给了三位科学家,以表彰他们对于DNA修复的机理研究。获奖者分别是来自瑞典的Tomas Lindahl、美国的Paul Modrich和土耳其的Aziz Sancar。其中,Tomas Lindahl是中     

Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

The use of reporter systems to analyze DNA double-strand break(DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination(HR),non-homologous end joining(NHEJ) and single-strand annealing(SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce I recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by l-Sce I could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction(qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseIV(lig4) when the NHEJ construct was cut by I-Sce I in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.     

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

The key residue for SSB-RecO interaction is dispensable for Deinococcus radiodurans DNA repair in vivo

The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein （SSB） coated single-stranded DNA （ssDNA）. However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO （drRecO-R121） as the key residue for RecO-SSB interaction. The substitution of drRecO-R121 with alanine greatly abolished the binding of RecO to SSB but not the binding to RecR. Meanwhile, SSB-coated ssDNA annealing activity was also compromised by the mutation of the residue of drRecO. However, the drRecO-R121A strain showed only modest sensitivity to DNA damaging agents. Taking these data together, arginine-121 of drRecO is the key residue for SSB-RecO interaction, which may not play a vital role in the SSB displacement and RecA loading process of RecFOR DNA repair pathway in vivo.     

DNA损伤修复机制——解读2015年诺贝尔化学奖

Tomas Lindahl, Paul Modrich和Aziz Sancar三位科学家因发现“DNA损伤修复机制”获得了2015年诺贝尔化学奖．Lindahl首次发现Escherichia Coli中参与碱基切除修复的第一个蛋白质--尿嘧啶 DNA糖基化酶（UNG）; Modrich重建了错配修复的体外系统,从大肠杆菌到哺乳动物深入探究了错配修复的机制; Sancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复的关键步骤,阐述了核苷酸切除修复的分子机制．DNA损伤是由生物所处体外环境和体内因素共同导致的,面对不同种类的损伤,机体启动多种不同的修复机制修复损伤,保护基因组稳定性．这些修复机制包括：光修复(light repairing);核苷酸切除修复（nucleotide excision repair, NER）;碱基切除修复（base excision repair, BER）;错配修复（mismatch repair, MMR）;以及DNA双链断裂修复（DNA double strand breaks repair, DSBR）．其中DNA双链断裂修复又分同源重组（homologous recombination, HR）和非同源末端连接（non homologous end joining, NHEJ）两种方式．本文将对上述几种修复的机制进行总结与讨论．     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Fast repair of dAMP hydroxyl radical adduct by verbascoside via electron transfer

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very impotant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activity and its mechanism of verbaseoside, isolated from Pedicularis species, towards dAMP-OH·was studied with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mmol/L dAMP aqueous solution containing verbascoside, the transient absorption spectrum of the hydroxyl adduct of dAMP decayed with the formation of that of the phenoxyl radical of verbascoside well under 100 microseconds after electron pulse irradiation. The result indicated that dAMP hydroxyl adducts can be repaired by verbascoside. The rate constants of the repair reaction was deduced to be 5.9×10~8 dm~3·mol~(-1)·s~(-1). A deeper understanding of this new repair mechanism will undo     

Polyomavirus interaction with the DNA damage response

Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication—normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response(DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated(ATM) and ATM- and Rad3-related(ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATMand ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.     

Knockouts of RecA-Like Proteins RadC1 and RadC2 Have Distinct Responses to DNA Damage Agents in Sulfolobus islandicus

RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we performed phylogenetic and genetic analysis of two RadC proteins from Sulfolobus islandicus. RadC is closer to the KaiC lineage of cyanobacteria and proteobacteria than to the lineage of the recombinases （RecA, RadA, and Rad51） and the recombinase paralogs （e.g., RadB, Rad55, and Rad51B）. Using the recently- established S. islandicus genetic system, we constructed deletion and over-expression strains of radC1 and radC2. Deletion of radC1 rendered the cells more sensitive to DNA damaging agents, methyl methanesulfonate （MMS）, hydroxyurea （HU）, and ultraviolet （UV） radiation, than the wild type, and a AradCIAradC2 double deletion strain was more sensitive to cisplatin and MMS than the AradC1 single deletion mutant. In addition, ectopic expression of His-tagged RadC 1 revealed that RadC I was co-purified with a putative structure-specific nuclease and ATPase, which is highly conserved in archaea. Our results indicate that both RadCI and RadC2 are involved in DNA repair. RadCl may play a general or primary role in DNA repair, while RadC2 plays a role in DNA repair in response to specific DNA damages.