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Ugly Rad23

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Last Updated: 02 July 2021

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General | Latest Info

Nucleotide excision repair is a major repair system for removal of DNA lesions induced by UV light and numerous chemical agents. The Cut-and-patch-type reaction mechanism involves concerted action of more than 25 proteins, sequentially implicated in recognition of DNA damage, unwinding of DNA around lesion, excision of single-strand piece of DNA containing damage, and subsequent gap filling DNA synthesis and ligation. NER consists of two subpathways. Genome-wide repair is taken care of by the global genome NER process, acting irrespective of genomic location of lesion or cell cycle stage. However, some lesions are repaired less efficiently by GG-NER. To prevent such lesions from obstructing the vital process of transcription for too long, transcription-couple NER subpathway acts as a fast backup system for clearing template strands of actively transcribe genes. Defective NER is associated with three clinically and genetically heterogeneous human syndromes: xeroderma pigmentosum, cockayne syndrome, and trichothiodystrophy. Patients suffering from XP exhibit severe sensitivity to sunlight, ocular symptoms, and cutaneous abnormalities, including very strong predisposition to develop skin cancer. Most XP patients carry defects in GG-NER and TC-NER, but only the GG-NER pathway is affected in XP-C patients. The XPC protein is essential for GG-NER of various types of DNA damage and is found in a tight complex with hHR23B, one of two human homologs of Saccharomyces cerevisiae DNA repair protein RAD23. HHR23B stimulates repair activity of XPC in in vitro reconstitution assays with recombinant proteins. A 56-amino-acid segment with predicted helical amphipathic structure containing XPC-binding domain of hHR23B appears sufficient for XPC stimulation. This suggests that hHR23B has structural rather than catalytic function. The vast majority of XPC protein is bind to hHR23B in vivo. However, in vitro, hHR23A, second human homolog of RAD23, can substitute for hHR23B in binding and stimulating XPC. This opens the possibility that both proteins are functionally interchangeable to some extent. The XPC-hHR23B complex has been identified as the primary DNA damage sensor that initiates GG-NER and has been shown to interact directly with essential repair and transcription factor TFIIH in vivo and in vitro. The XPC-hHR23B complex senses different types of damage based on disrupted base pairing and stimulates association of TFIIH with damage DNA in cell extracts. After initial subpathway-specific lesion detection, XPB and XPD helicase subunits of TFIIH open DNA helix around lesion. XPA together with heterotrimeric replication protein may function as a common damage verifier before DNA is incise on both sides of injury by XPG and ERCC1 / XPF endonucleases. In addition to the XPC-binding domain, S. Cerevisiae and mammalian RAD23 proteins harbor amino-terminal ubiquitin-like moiety and two so-call ubiquitin-associate domains. In S. Cerevisiae, UbL domain is indispensable for repair function of RAD23.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Introduction

Each day, endogenous and environmental assaults generate > 10 4 DNA lesions in any given cell, which are corrected by DNA repair pathways responsible for maintenance of DNA integrity. Global genome-nucleotide excision repair is a generic pathway that repairs bulky DNA lesions such as UV-induced thymidine dimers or cisplatin-DNA adducts. DNA damage recognition and GG-NER are initiated by xeroderma pigmentosum complementation group C protein, which functions as a complex with hRad23B and centrin2 to recognize DNA helix distortions. XPC-centrin2 interaction is mediated by binding of COOH-terminal domain of centrin2 to W1L4L8 motif of XPC. Recruitment of XPC / hRad23B / centrin2 complex is rate-limiting step in GG-NER and is dependent on XPC abundance and stability. On binding to DNA, XPC / hRad23B / centrin2 complex recruits DNA helicases XPD and XPB, which unwind DNA to allow downstream repair proteins of pathway to localize to lesion. Autosomal recessive defects in this DNA repair process result in xeroderma pigmentosum in which patients carrying mutations in key GG-NER components, including XPC, develop severe UV sensitivity, trichothiodystrophy, neural defects, and elevated risk of cancer. In addition to repair of DNA lesions, maintenance of genomic integrity is also dependent on exquisite regulation of equal chromosomal segregation during cell division. To form bipolar microtubule-base mitotic spindle, centrosome duplicates once in each cell cycle to give rise to two mitotic spindle poles. In many cancers, disruption of this process leads to centrosome amplification characterized by multiple centrosomes, increased accumulation of pericentriolar material, and / or supernumerary centrioles, which ultimately lead to chromosomal instability and aneuploidy. Centrin is a 20-kDa cytosolic calcium-binding regulatory protein required for centrosome homeostasis. Humans and mice have four centrin genes. Cetn1 is expressed in male germ cells, certain neurons, and terminally differentiate ciliated cells; Cetn2 and Cetn3 are expressed in all somatic cells; and Cetn4 is expressed in terminally differentiate ciliated cells. In mammalian cells, centrin2 is distributed diffusely in cytoplasm with prominent centriole localization and also sporadically in the nucleus. Centrin2 binds diverse centrosome proteins, but direct binding of centrin2 to DNA repair protein XPC and variable nuclear localization of centrin2 lead us to investigate cellular consequences of interaction between centrin2 and XPC in more detail. Studies reported here compare responses of two different human breast cancer cell lines to DNA damage. One of theses cell lines, MCF-7, represents an early-stage breast cancer phenotype that has retained p53-mediate cell cycle checkpoint controls, whereas other cell line, MDA-MB 231, represents an advanced breast cancer phenotype with aggressive metastatic properties, loss of p53-mediate checkpoint control, and high degree of genomic instability.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

MATERIALS AND METHODS

Each day, endogenous and environmental assaults generate > 10 4 DNA lesions in any given cell, which are corrected by DNA repair pathways responsible for maintenance of DNA integrity. Global genome-nucleotide excision repair is a generic pathway that repairs bulky DNA lesions such as UV-induced thymidine dimers or cisplatin-DNA adducts. DNA damage recognition and GG-NER are initiated by xeroderma pigmentosum complementation group C protein, which functions as a complex with hRad23B and centrin2 to recognize DNA helix distortions. XPC-centrin2 interaction is mediated by binding of COOH-terminal domain of centrin2 to W1L4L8 motif of XPC. Recruitment of XPC / hRad23B / centrin2 complex is rate-limiting step in GG-NER and is dependent on XPC abundance and stability. On binding to DNA, XPC / hRad23B / centrin2 complex recruits DNA helicases XPD and XPB, which unwind DNA to allow downstream repair proteins of pathway to localize to lesion. Autosomal recessive defects in this DNA repair process result in xeroderma pigmentosum in which patients carrying mutations in key GG-NER components, including XPC, develop severe UV sensitivity, trichothiodystrophy, neural defects, and elevated risk of cancer. In addition to repair of DNA lesions, maintenance of genomic integrity is also dependent on exquisite regulation of equal chromosomal segregation during cell division. To form bipolar microtubule-base mitotic spindle, centrosome duplicates once in each cell cycle to give rise to two mitotic spindle poles. In many cancers, disruption of this process leads to centrosome amplification characterized by multiple centrosomes, increased accumulation of pericentriolar material, and / or supernumerary centrioles, which ultimately lead to chromosomal instability and aneuploidy. Centrin is a 20-kDa cytosolic calcium-binding regulatory protein required for centrosome homeostasis. Humans and mice have four centrin genes. Cetn1 is expressed in male germ cells, certain neurons, and terminally differentiate ciliated cells; Cetn2 and Cetn3 are expressed in all somatic cells; and Cetn4 is expressed in terminally differentiate ciliated cells. In mammalian cells, centrin2 is distributed diffusely in cytoplasm with prominent centriole localization and also sporadically in the nucleus. Centrin2 binds diverse centrosome proteins, but direct binding of centrin2 to DNA repair protein XPC and variable nuclear localization of centrin2 lead us to investigate cellular consequences of interaction between centrin2 and XPC in more detail. Studies reported here compare responses of two different human breast cancer cell lines to DNA damage. One of theses cell lines, MCF-7, represents an early-stage breast cancer phenotype that has retained p53-mediate cell cycle checkpoint controls, whereas other cell line, MDA-MB 231, represents an advanced breast cancer phenotype with aggressive metastatic properties, loss of p53-mediate checkpoint control, and high degree of genomic instability.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

RESULTS

Target disruption of mouse mHR23B gene. Mouse mHR23B locus was isolated and partly characterize. Deletion of exon III not only removes sequence encoding amino acid residues 148 to 228 of mHR23B protein but also results in frameshift and accordingly is expected to give rise to null allele. Following transfection of ES cell line E14, properly targeted heterozygous ES cells were obtained at a frequency of 21 %. Two independent ES clones for which absence of visible chromosomal abnormalities and additional randomly integrated constructs had been verified were used for blastocyst injections. Germ line transmission was obtained for both clones. Heterozygous offspring from matings between chimeric males and C57BL / 6 female mice were intercrossed in order to generate homozygous mutant mHR23B animals. In parallel, these matings serve to isolate MEFs of different genotypes from day 13. 5 embryos. The Effect of targeted disruption on expression of mHR23B gene was analyzed in MEFs: neither mHR23B mRNA nor mHR23B protein could be detected in mHR23B / cells by RNA and immunoblot analyses, respectively. We conclude that we have created mHR23B null mutants. Homozygous mouse mutants and cell lines from two independent ES transformants yield identical results in all subsequent studies, indicating that findings reported below are not due to uncontrolled events that might have occurred in one targeted ES clone but are the result of mHR23B inactivation. MHR23B-deficient cells are NER proficient. In view of the role of S. Cerevisiae RAD23 in NER and tight interaction of mHR23B with XPC, we examined cellular survival of wild-type, heterozygous, and homozygous mHR23B mutant MEFs after exposure to increasing UV doses. Unexpectedly, UV survival of mHR23B + / and mHR23B / cells appear indistinguishable from that of wild-type cells. Moreover, mHR23B-deficient MEFs show normal DNA repair synthesis and recovery of RNA synthesis after UV exposure, indicating that neither GG-NER nor TC-NER subpathways were affect. Also, in other respects, mHR23B-deficient MEFs behave normally. Assuming that total mHR23 inactivation would result in DNA repair deficiency, as in S. Cerevisiae, these data suggest that mHR23A can fully substitute for mHR23B, at least for its function in NER, not only in vitro but also in vivo. MHR23B deficiency causes impaired embryonic development and intrauterine death. When heterozygous animals were crossed to produce mHR23B-deficient mice, target mHR23B allele was found to segregate at a ratio far below that expected by Mendelian inheritance, suggesting that lack of mHR23B protein causes intrauterine and / or perinatal death. Since mHR23B-deficient MEF lines were obtained at almost Mendelian ratio, lethal events must occur after day 13. 5 of gestation. Analysis of large series of embryos at different stages of development revealed a near twofold reduction in the number of viable mHR23B-deficient embryos between E13. 5 and E15.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

DISCUSSION

MHR23 deficiency does not result in NER defect. RAD23 mutants are unique among S. Cerevisiae NER mutants in several respects. Despite intermediate UV sensitivity, suggesting partial NER impairment, deletion mutants of both S. Cerevisiae and Schizosaccharomyces pombe do not display, paradoxically, detectable global genome and transcription-couple repair, indicating that NER is completely disturbed. Within NER machinery, RAD23 is the only component with multiple connections with ubiquitin system. The RAD23 protein has an ubiquitin-like N terminus that is essential for its function in repair in vivo. Two ubiquitin-associate domains within protein are very strongly conserve, which underlines their functional importance. Protein interacts physically via its ubiquitin-like domain with components of 26S proteasome and inhibits multiubiquitination in vitro. In view of its strong interaction with RAD4, it is likely that the main role of RAD23 in NER is mediate via this repair component. Remarkably, S. Cerevisiae RAD4 and RAD23 appear to be involved in both NER subpathways, whereas the mammalian counterpart of RAD4, XPC, is involved only in GG-NER. Of the two homologs of RAD23 in mammals, hHR23B is the main partner of XPC. XPC-hHR23B heterodimer is identified as the first initiator of damage recognition in global genome repair, and it is also found to stimulate XPC in in vitro NER. In this study, we have analyzed the function of mHR23B in vivo by generating mHR23B knockout mouse model. Surprisingly, in contrast to S. Cerevisiae, no apparent NER phenotype is detected in mHR23B-deficient cells, which are not UV sensitive and show efficient global genome repair and transcription-couple repair. These data demonstrate that NER, and in particular the function of XPC, is not significantly affected by the absence of mHR23B. Assuming that as in S. Cerevisiae, mammalian RAD23 homologs are important for NER, most plausible interpretation for our findings is that in the absence of mHR23B, mHR23A protein can functionally replace it, including binding and stimulation of XPC. This is consistent with in vitro redundancy between human HR23 homologs in NER and recent identification of XPC-hHR23A subcomplex in whole-cell extracts. These findings support the in vivo function of HR23A in GG-NER. Thus, they argue against the model that HR23A protein specifically interacts with hitherto unidentified second RAD4-like homolog in mammals specific for TC-NER, so that both together would cover function of single RAD4 and RAD23 genes in S. Cerevisiae. However, it remains puzzling why HR23B protein is normally predominantly associated with XPC in living cells, whereas this study suggests that HR23A appears to be equally able to perform this function in NER. Therefore, functional distinction between HR23A and HR23B proteins is still unresolved. Mice lacking mHR23B display severe, unexpected phenotype. Whereas apparent NER defect could not be detect, mHR23B-deficient mice exhibit severe phenotype, which is quite different from abnormalities observed in mouse models for other NER genes.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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