How is damaged dna detected

Intact DNA from the sister chromatid or homologous chromosome shown in grey lines is used as a template to replace the genetic information lost during the nucleolytic process. Following the nucleolytic process, Holliday junction, branch migration, nuclease resolution of the junction and ligation of the DNA complete the recombinational repair b Non-homologous end-joining pathway.

In the latter case, the ends are processed resulting in the loss or addition of nucleotides and is frequently observed in mammalian cells. Other factors that may be required for this process remain to be identified. Rad50 and Mre11 proteins share sequence similarity with E. Rad50, like SbcC, is a coiled-coil protein with ATP binding motifs and shares sequence similarity with the structural maintenance of chromosome SMC family of proteins.

This protein family is implicated in chromosome condensation and segregation, transcriptional repression and recombination Connelly et al. Mre11, like SbcD, belongs to a family of phosphoesterases Sharples and Leach, The specific involvement of a helicase in DSB processing reaction remains to be addressed. Studies of mre11 mutants indicate that this protein and presumably the other members of the complex has both enzymatic and structural functions. Nuclease-deficient mre11 mutant is not as sensitive to IR as the null mutants but has normal telomere length, suggesting that the Mre11 nuclease activities are not required for chromosome end protection and may not be as crucial in mitosis as in meiosis Moreau et al.

The exonuclease activity of Mre11 is enhanced by Rad50 Paull and Gellert, More studies are needed to explain how NBS1 modulates the activities of Mre These processes are coupled with DNA synthesis to replace the genetic information eliminated during nucleolytic processing of the DSB.

Subsequently, Holliday junctions are formed and heteroduplex regions are extended by branch migration of the Holliday junction. Finally, resolution of the Holliday junctions yields mature recombinants with or without crossover in relation to the flanking markers see Figure 2. These genes can be divided into three subgroups. RAD52 is the gene required for all HR pathways and thus rad52 mutants have the most severe phenotype among all mutants of the RAD52 epistasis group Paques and Haber, As described above, mre11, rad50 and xrs2 mutants share similar phenotypes and these genes form the third subgroup.

Biochemical studies have provided a framework for the HR pathways mediated by these proteins. Eukaryotic Rad51 protein, a homologue of Escherichia coli RecA protein, plays a central role in HR and recombinational repair.

Similar to E. However, extensive strand exchange was not observed under standard conditions, indicating the requirement of additional proteins. Although yeast rad51 mutants are viable, knockout of the mouse Rad51 gene leads to embryonic lethality Lim and Hasty, ; Tsuzuki et al. Using a tetracyclin-regulated promoter to control the expression of Rad51, Sonoda et al.

Furthermore, sister chromatid exchange frequency is greatly reduced in cells lacking Rad51, suggesting a potential role for HR in sister chromatid exchange Sonoda et al.

The identification of novel Rad51 interaction proteins see below as well as the discoveries of multiple cellular processes involving Rad51 will provide a better explanation for the lethal phenotype caused by Rad51 inactivation in mammals. Both genetic and biochemical data indicate that Rad52 interacts with the Rad51 recombinase Baumann and West, b.

Recently, human Rad52 was shown to bind directly to DSBs and, like Ku see below , protect DNA from exonuclease attack and promote end-to-end association through intermolecular interactions between hRad52 Van Dyck et al.

Surprisingly, while both biochemical and cellular studies suggest an important role for Rad52 in HR in yeast, knockout of Rad52 results in a subtle phenotype in the mouse. This raises the possibility that either mammalian Rad52 function may not be similar to that of ScRad52 or that there may be yet to be identified homologs of RAD52 in the mammalian genome. Rad51 was shown to interact with Rad54 and this interaction is functionally important for HR Clever et al.

The important role of Rad54 in HR and sister chromatid exchange has been demonstrated by gene inactivation in both mouse and chicken cells Essers et al. In mammals, additional proteins are required to facilitate the error-free repair process see below. Recent studies demonstrated that NHEJ activities can be re-constituted in vitro with two separate chromatographic fractions Baumann and West, a.

Ku was first identified as an autoimmune antigen in scleroderma-polymyositis and other autoimmune patients. Ku binds to DNA avidly in a sequence-independent fashion. Furthermore, Ku interacts with Sir4 which is involved in the transcriptional silencing of telomere-adjacent region Tsukamoto et al.

These biochemical properties of Ku likely underlie Ku's role in telomere maintenance and transcriptional silencing that was first predicted based on genetic studies in yeast. Recent studies of DNA-PK structure by electron crystallography have revealed an open channel and an enclosed cavity with openings that allow entry of ss DNA.

Guided by the results, further biochemical studies suggested that activation of the kinase requires both ds and ss DNA Leuther et al. However, functions such as growth regulation and telomere maintenance appear to be uniquely involving Ku.

This was confirmed in the mouse knockout model in which the ligase IV gene had been inactivated Grawunder et al. The biochemical properties and function of these tumor suppressors are described below.

A-T cells display several abnormalities in the repair of DSBs; i higher levels spontaneous intra-chromosomal recombination after IR; ii frequent error-prone intra- and extra-chromosomal recombinations; iii aberrant NHEJ characterized by large deletions or insertions at the site of joining Meyn, ; see Shiloh, ; and references therein.

Since the studies described above were performed in transformed A-T cell lines, it is possible that DSB-induced signaling pathways might be de-regulated in these cell lines. A similar observation was made in yeast, suggesting that RPA phosphorylation in response to DSB is conserved in eukaryotes Brush et al.

Recently, tyrosine phosphorylation of Rad51 was found to be dependent on c-Abl kinase both in vitro and in vivo Chen et al. Yuan et al. In contrast, Chen et al. Since phosphorylation of Rad51 by c-Abl enhances its interaction with Rad52 and since IR-induced enhancement of tyrosine phosphorylation of Rad51 was not seen in A-T cells, Chen et al. Although these studies provide a plausible mechanism for ATM involvement in DSBR, how the post-translational modification of Rad51 affects its function during HR in vivo remains to be established.

IR-induced phosphorylation of these proteins is minimal in A-T cells Zhao et al. BRCA1 and BRCA2 are two tumor suppressor genes mutated in a significant percentage of patients with familial breast cancer reviewed by Koller, this issue. Insights into the function of these two proteins are based on studies of knockout phenotypes and identification of proteins that interact with them. These studies indicate that both genes might be involved in DSBR.

BRCA1 has been implicated in the regulation of replication checkpoint Scully et al. Future studies of how DSBR pathways are affected in these cells will provide important information on how these newly evolved tumor suppressors modulate DSBR.

Telomeres are special structures at the ends of chromosomes Griffith et al. Telomeres protect chromosome ends from DNA degradation and fusion between chromosome ends. Ku-deletion mutants display telomere shortening Boulton and Jackson, , loss of telomere silencing and dissociation of telomere from the nuclear periphery while sir mutants are mainly defective in telomere silencing Boulton and Jackson, ; Laroche et al.

Both ku and sir mutants are defective in NHEJ. Recent studies demonstrated that in the presence of even a single DSB, Sir and Ku proteins will be partially dissociated from telomere and are diffusely distributed within the nucleus.

Interestingly, the re-localization of Ku and Sir requires the checkpoint proteins Rad9 and Mec1 Martin et al. Unlike ku or sir mutants, rad50, mre11 , or xrs2 mutants have shortened telomeres without the loss of telomere silencing.

Taken together, a number of DSBR proteins are also involved in telomere maintenance through interaction with different telomere binding proteins. HR processes have been extensively studied in the context of mitotic recombination, meiotic recombination, and mating type switching Nickoloff and Hoekstra, ; Paques and Haber, ; Petukhova et al. Earlier studies of hamster mutant cell lines led to the identification of important genes in NHEJ. Only recently, genes important for HR were identified.

The role of HR in maintaining the genomic stability in mammals is largely unknown. In addition to a repair role, HR activities may also contribute to the loss of heterozygosity Moynahan and Jasin, ; Shulman et al. Increasing evidence also indicates the role for HR in sister chromatid recombination Sonoda et al. In summary, emerging evidence indicates broader roles for HR pathways in maintaining genomic stability, but the details of most of the processes remain to be elucidated.

Thus it is likely that HR is a preferred mechanism for repairing DSBs at specific stages of the cell cycle. Since it has been hypothesized that Rad52 and Ku are involved in the choice of repair pathways Van Dyck et al. Whether there is a link between DNA repair components and checkpoint molecules leading to specific checkpoint or repair defects also needs to be explored.

While mouse models of defective nucleotide excision repair and mismatch repair have provided crucial information regarding the effects of repair defects on development and tumorigenesis, it is only recently that such models for DSBR were established. We will summarize findings from these mouse models Table 1. Mutant ES cells heterozygous for Mre11 disruption have no apparent growth abnormalities in culture.

However, homozygous mutant ES cells are non-viable, suggesting that mammalian Mre11 is an essential gene Xiao and Weaver, Similarly, no ES cells homozygous for targeted disruption of Rad50 can be isolated. Homozygous mutations of Rad50 in mice result in early embryonic lethality at E6.

Therefore, mutations in Mre11 and Rad50 cause more severe phenotypes in mammals than in yeast. Since RAD50 and MRE11 are not essential genes in yeast, mammalian homologs may have evolved to carry out additional functions.

One possibility is that the interaction between this protein complex and recently evolved gene products, such as BRCA1 that plays important roles in both DNA repair and cell cycle checkpoints. All NBS patients studied to date have mutations resulting in the truncation of the NBS1 gene, mostly at the amino terminus Varon et al.

Mre11 nuclease activities are enhanced by the presence of Rad50 although the combination of the three components results in the highest activities Paull and Gellert, Homozygous Rad51 mutation leads to early embryonic lethality associated with decreased proliferation in mouse embryos. Fibroblasts derived from double mutant embryos fail to proliferate in tissue culture Lim and Hasty, The mild phenotype exhibited by mice deficient in Rad52 may be explained by the existence of additional Rad52 homologs.

Rad54 null mice are viable but surprisingly, have no apparent defects in spermatogenesis and oogenesis Essers et al. This raises the possibility of the existence of homologs of Rad54 that may function specifically during meiosis. There is a meiosis-specific Rad51 homolog, Dmc1, which is specifically expressed in testis Yoshida et al. Homozygous deletion of Dmc1 in the mouse results in the failure of homologous chromosome synapsis during meiosis Pittman et al.

Readers are referred to a recent review for a more detailed description and comparison of mutant mice deficient for different components of the DNA-PK Smith and Jackson, The V D J recombination defect in these mutant mice lies in the joining step. Contrary to expectations, both hairpin coding ends and blunt full-length signal ends accumulate in the absence of Ku, indicating that Ku is not required to protect V D J recombination intermediates Zhu et al.

In Ku80 and Ku70 mutant mice, both coding-end joining and signal-end joining are severely impaired Nussenzweig et al. Recent studies have indicated that immunoglobulin heavy chain class switching, which involves the processing of DSB intermediates Wuerffel et al. Moreover, Ku has been shown to participate in the sequence-specific transcriptional repression of a specific promoter and it appears that DNA-PKcs is required for such repression Giffin et al.

Ku and Ku deficiency results in growth defects Nussenzweig et al. Mice deficient in Ku70 undergo T cell development but form lymphomas. Taken together, mice carrying a deletion of an individual component of the DNA-PK complex provide new insights into their shared and distinct functions and lay the foundation of future mechanistic studies.

Lymphocyte development is blocked and both coding-end and signal-end joining in V D J recombination are defective in these mice Frank et al. Embryonic fibroblasts MEFs derived from these mice proliferate poorly, enter senescence prematurely, and are extremely sensitive to IR Frank et al.

All these phenotypes, except the embryonic lethality, are similar to those seen in Ku-deficient cells, suggesting that Ku, XRCC4 and ligase IV may be involved in the same end-joining process. Thomas, Lodish, H. Molecular Biology of the Cell , 5th ed. New York, Freeman, Sinha, R. Photochemical and Photobiological Sciences 1 , — Restriction Enzymes. Genetic Mutation. Functions and Utility of Alu Jumping Genes. Transposons: The Jumping Genes.

DNA Transcription. What is a Gene? Colinearity and Transcription Units. Copy Number Variation. Copy Number Variation and Genetic Disease. Copy Number Variation and Human Disease.

Tandem Repeats and Morphological Variation. Chemical Structure of RNA. Eukaryotic Genome Complexity. RNA Functions. Citation: Clancy, S. Nature Education 1 1 DNA integrity is always under attack from environmental agents like skin cancer-causing UV rays. Aa Aa Aa. Table 2.

Figure Detail. Figure 1. Figure 3. Figure 2. Additional DNA Repair mechanisms. Figure 4. References and Recommended Reading Branze, D.

Thomas, Lodish, H. New York, Freeman, Sinha, R. Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article.

Save Cancel. Flag Inappropriate The Content is: Objectionable. Flag Content Cancel. Therefore, one can reasonably assume existence of specialized molecular mechanisms, which assist XPC to discriminate its rare target sites efficiently from intact DNA present in large excess. Its binding to pyrimidine 6—4 pyrimidone photoproducts 6—4PPs is especially strong, whereas CPDs show moderate but significant affinity [ 27 , 28 ].

In contrast, bulky base adducts induced by chemical mutagens are relatively poor substrates [ 29 , 30 ]. Biochemical studies have shown that DNA lesions within the nucleosome core tend to refrain from interaction with XPC and the following repair reaction [ 37 , 38 ].

Because 6—4PPs are shown to be generated by UV irradiation in chromatin in a relatively random manner [ 46 ], this fact strongly suggests existence of unprecedented molecular mechanisms that enable alteration of chromatin structures prior to lesion detection by XPC. We have recently reported that XPC physically interacts with histone H3 and this interaction is negatively regulated by acetylation of the histone protein [ 47 ].

Based on these findings, we propose that DNA damage associated with a relatively large helix distortion may be able to induce local reorganization of chromatin including deacetylation of histones, which may contribute to efficient recruitment of XPC [ 47 ].

Since chemical changes in DNA caused by lesions per se are only small, roles for chromatin structures could be completely different from the situation of UV-DDB-bound lesions.

In order to explore novel molecular mechanisms underlying regulation of DNA damage recognition for GG-NER, we have recently set up a confocal laser scanning microscopy equipped with a nm femtosecond fiber laser, with which local DNA damage similar to that induced by nm UV irradiation can be generated within cell nuclei by a principle of three-photon absorption.

This system enables us to observe retarded accumulation of fluorescence-tagged XPC to the damaged sites after suppression of DDB2 expression or treatment with a histone deacetylase inhibitor Fig. DNA damage was induced with the nm femtosecond laser and three-photon absorption within subnuclear regions of human osteosarcoma U2OS cells stably expressing mCherry-fused XPC. Mean values and standard deviations were calculated from 10 a and 17 b samples, respectively.

Utilizing intrinsic sites that allow XPC binding, it is possible that this stepwise strategy further extends potential of lesion recognition in GG-NER. One of our goals would be to recapitulate the GG-NER processes with damaged chromatin substrates in the cell-free system.

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Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase.