Comparison of the Global Genomic and Transcription-Coupled Repair Rates of Different Lesions in Human Cells

There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences. There are data that GGR rates depend on the chemical nature of the lesions in a manner that the lesions inflicting larger distortion on the DNA double helix are repaired at higher rate. It is not known whether the TCR repair rates depend on the type of lesions and in what way. To address this question human cells were transfected with pEGFP and pEYFP plasmids treated with UV light, cis-diamminedichloroplatinum(II) (cisplatin) and angelicin and 24 h later the restored fluorescence was measured and used to calculate the respective NER rates. In a parallel series of experiments the same plasmids were incubated in repair-competent protein extracts to determine GGR rates in the absence of transcription. From the two sets of data, the TCR rates were calculated. We found out that cisplatin, UV light and angelicin lesions were repaired by GGR with different efficiency, which corresponded to the degree of DNA helix distortion induced by these agents. On the other hand the three lesions were repaired by TCR at very similar rates which showed that TCR efficiency was not directly connected with the chemical nature of the lesions.

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USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.663411 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ying Zhang ◽

Imke K. Mandemaker ◽

Syota Matsumoto ◽

Oded Foreman ◽

Christopher P. Holland ◽

...

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Uv Light ◽

Double Helix ◽

Dna Lesions ◽

Physiological Importance ◽

Ubiquitin E3 Ligase ◽

Pyrimidine Dimers ◽

Nucleotide Excision ◽

Dna Double Helix

Nucleotide excision repair (NER) is a pathway involved in the repair of a variety of potentially mutagenic lesions that distort the DNA double helix. The ubiquitin E3-ligase complex UV-DDB is required for the recognition and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) lesions through NER. DDB2 directly binds CPDs and subsequently undergoes ubiquitination and proteasomal degradation. DDB2 must remain on damaged chromatin, however, for sufficient time to recruit and hand-off lesions to XPC, a factor essential in the assembly of downstream repair components. Here we show that the tumor suppressor USP44 directly deubiquitinates DDB2 to prevent its premature degradation and is selectively required for CPD repair. Cells lacking USP44 have impaired DDB2 accumulation on DNA lesions with subsequent defects in XPC retention. The physiological importance of this mechanism is evident in that mice lacking Usp44 are prone to tumors induced by NER lesions introduced by DMBA or UV light. These data reveal the requirement for highly regulated ubiquitin addition and removal in the recognition and repair of helix-distorting DNA damage and identify another mechanism by which USP44 protects genomic integrity and prevents tumors.

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Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

Molecular and Cellular Biology ◽

10.1128/mcb.5.2.398 ◽

1985 ◽

Vol 5 (2) ◽

pp. 398-405 ◽

Cited By ~ 28

Author(s):

J S Rubin ◽

V R Prideaux ◽

H F Willard ◽

A M Dulhanty ◽

G F Whitmore ◽

...

Keyword(s):

Dna Repair ◽

Dna Sequences ◽

Chromosomal Localization ◽

Excision Repair ◽

Repair Process ◽

Human Cells ◽

Gene Products ◽

Repair Gene ◽

Human Dna ◽

Dna Repair Gene

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.

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Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: Analogous measurements in insect and human cells

Cell ◽

10.1016/0092-8674(80)90440-7 ◽

1980 ◽

Vol 21 (3) ◽

pp. 773-783 ◽

Cited By ~ 243

Author(s):

R Sinden

Keyword(s):

Double Helix ◽

Human Cells ◽

E Coli ◽

Dna Double Helix

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Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability

Genes ◽

10.3390/genes10020170 ◽

2019 ◽

Vol 10 (2) ◽

pp. 170 ◽

Cited By ~ 20

Author(s):

Arindam Datta ◽

Robert M. Brosh Jr.

Keyword(s):

Dna Damage ◽

Fanconi Anemia ◽

Excision Repair ◽

Double Helix ◽

Bone Marrow Failure ◽

Molecular Genetic ◽

Replication Stress ◽

Gene Products ◽

Hematopoietic Stem ◽

Dna Double Helix

Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA.

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Excision-repair patch lengths are similar for transcription-coupled repair and global genome repair in UV-irradiated human cells

Mutation Research/DNA Repair ◽

10.1016/s0921-8777(97)00029-3 ◽

1997 ◽

Vol 385 (2) ◽

pp. 95-105 ◽

Cited By ~ 25

Author(s):

Krista K Bowman ◽

Charles Allen Smith ◽

Philip C Hanawalt

Keyword(s):

Excision Repair ◽

Human Cells ◽

Repair Patch ◽

Global Genome Repair ◽

Transcription Coupled Repair ◽

Genome Repair

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Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

Nucleic Acids Research ◽

10.1093/nar/gkz645 ◽

2019 ◽

Vol 47 (18) ◽

pp. e109-e109

Author(s):

Eliraz Greenberg ◽

Hodaya Hochberg-Laufer ◽

Shalev Blanga ◽

Noa Kinor ◽

Yaron Shav-Tal

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Dna Sequences ◽

Plasmid Dna ◽

Double Helix ◽

Rna Molecules ◽

Rna Fish ◽

Dna Double Helix ◽

Intracellular Detection

Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.

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ATP-Dependent Chromatin Remodeling by the Cockayne Syndrome B DNA Repair-Transcription-Coupling Factor

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7643-7653.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7643-7653 ◽

Cited By ~ 246

Author(s):

Elisabetta Citterio ◽

Vincent Van Den Boom ◽

Gavin Schnitzler ◽

Roland Kanaar ◽

Edgar Bonte ◽

...

Keyword(s):

Dna Repair ◽

Chromatin Remodeling ◽

Chromatin Structure ◽

Excision Repair ◽

Double Helix ◽

Coupling Factor ◽

Cockayne Syndrome ◽

Direct Role ◽

Dna Double Helix

ABSTRACT The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

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Sequence-specific assignments and their use in NMR studies of DNA structure

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500004212 ◽

1987 ◽

Vol 20 (1-2) ◽

pp. 1-34 ◽

Cited By ~ 77

Author(s):

B. R. Reid

Keyword(s):

Dna Sequences ◽

Nearest Neighbor ◽

Context Effects ◽

Double Helix ◽

Dna Structure ◽

Nmr Studies ◽

Specific Sequence ◽

X Ray ◽

Fibre Diffraction ◽

Number Of Publications

There has been a surge of recent interest, reflected by a sharp increase in the number of publications, in the area of high-resolution nuclear magnetic resonance (NMR) studies of DNA. The goal of many of these studies is to monitor the structure of biologically important DNA sequences directly in solution; the impetus for such studies was the realization, from early single-crystal X-ray structures, that nearest-neighbor context effects are a major determinant of local structure in short double-helical DNAs (Dickerson & Drew, 1981; Dickerson, 1983). Thus, instead of the previously assumed regular averaged structure of the double helix derived from fibre diffraction analysis, the more interesting concept emerged that specific sequence-dependent distortions from ‘classical’ DNA structure might be responsible for the recognition of such sequences by a variety of ligands such as repressors, polymerases, drugs, etc.

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Higher Order Structures in Chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070278 ◽

1978 ◽

Vol 36 (3) ◽

pp. 545-556

Author(s):

H. Ris

Keyword(s):

Chelating Agents ◽

Chromosome Structure ◽

Double Helix ◽

The Other ◽

Interphase Chromatin ◽

Biochemical Studies ◽

Specific Fraction ◽

Dna Double Helix ◽

20 Nm ◽

Order Structures

Chromosomes are units of the genom in eukaryotes containing a specific fraction of the DNA characteristic for the species. This DNA forms the backbone of the chromosome and usually is represented by a single DNA double helix stretching from one end of the chromosome to the other. Relative to the size of most nuclei the length of the DNA is considerable. In humans for instance the haploid set of DNA is about 1 m long and in certain amphibia can measure many meters. The analysis of chromosome structure is mainly concerned with the various levels of folding or coiling by which this DNA is compacted in a regular way into chromosomes of interphase and mitotic stages. The electron microscope has played an important role in the discovery of chromosome organization. The 20 nm fiber was first described in 1956 as a unit of interphase chromatin (Ris, 1956). Later the 10 nm fiber was recognized as the component of chromatin dispersed in the presence of chelating agents for biochemical studies (Ris, 1961).

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