Biomedical subjects

G Dianov

Publications and source records attributed to G Dianov.

13 recordsLinked to original sources

PubMed2001

A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro.

Several recent studies have shown that human topoisomerase I (htopoI) can recognize various DNA lesions and thereby form a covalent topoisomerase I-DNA complex, which is known to be detrimental to cells. We have investigated whether htopoI recognizes another htopoI that is covalently trapped on a DNA substrate. For this purpose we created an artificial DNA substrate containing a specific topoisomerase I binding sequence, where the enzyme was trapped in the covalently bound form. We demonstrate that, in vitro, free htopoI stimulates the formation of an additional cleavage complex immediately upstream of the covalently bound topoisomerase I. The predominant distance between the two cleavage sites is 13 nt. In addition we find that these two enzymes may form direct protein-protein contacts and we propose that these may be mediated through the formation of a dimer by domain swapping involving the C-terminal and the core domains. Finally, we discuss the possibility that the double cleavage reaction may be the initial step for the removal of the recognized cleavage complex.

Animals↗

PubMed2001

DNA repair and mutagenesis in Werner syndrome.

Werner syndrome (WS) is the hallmark premature aging syndrome in which the patients appear much older than their actual chronological age. The disorder is associated with significantly increased genome instability and with transcriptional deficiencies. There has been some uncertainty about whether WS cells are defective in DNA repair. We thus examined repair in vitro in nuclear and mitochondrial DNA. Whereas cellular studies so far do not show significant DNA repair deficiencies, biochemical studies with the Werner protein clearly indicate that it plays a role in DNA repair.

Cell Line↗

PubMed1999

Repair of 8-oxoguanine in DNA is deficient in Cockayne syndrome group B cells.

The incision of the 8-oxoguanine in DNA by normal and Cockayne Syndrome (CS) cell extracts has been investigated. The incision in extracts derived from CS cells was approximately 50% of the incision level compared with extracts prepared from normal cells. In contrast, the incision rate of uracil and thymine glycol was not defective in CS cells. The deficiency in 8-oxoguanine incision was also demonstrated in a CS family. Whereas the proband had markedly less incision compared with the normal siblings, the parents had intermediate levels. The low level of 8-oxoguanine-DNA glycosylase in CS extracts correlates with the reduced expression of the 8-oxoguanine-DNA glycosylase gene (hOGG1) in CS cells. Both the levels of expression of the hOGG1 gene and the incision of 8-oxoguanine in DNAincreased markedly after transfection of CS-B cells with the CSB gene. We suggest that the CSB mutation leads to deficient transcription of the hOGG1 gene and thus to deficient repair of 8-oxoguanine in DNA.

Base Sequence↗

PubMed1998

Oxidative DNA damage processing and changes with aging.

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Aging↗

PubMed1998

Repair pathways for processing of 8-oxoguanine in DNA by mammalian cell extracts.

The repair pathways involved in the removal of 8-oxo-7, 8-dihydroguanine (8-oxoguanine) in DNA by mammalian cell extracts have been examined. Closed circular DNA constructs containing a single 8-oxoguanine at a defined site were used as substrates to determine the patch size generated after in vitro repair by mammalian cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that up to 75% of the 8-oxoguanine was repaired via the single nucleotide replacement mechanism in both human and mouse cell extracts. Approximately 25% of the 8-oxoguanine lesions were repaired by the long patch repair pathway. Repair incorporation 5' to the lesion, characteristic for nucleotide excision repair, was not significant. Elimination of the DNA polymerase beta (polbeta)-dependent single nucleotide base excision repair pathway in extracts prepared from polbeta-deficient mouse cells resulted in extension of the repair gap to 4-5 nucleotides 3' to the lesion in 50% of the repair events, suggesting the increased involvement of the long patch repair pathway. However, about one-half of the 8-oxoguanine repair was still accomplished through replacement of only one nucleotide in the polbeta-deficient cell extracts. These data indicate the existence of an alternative polbeta-independent single nucleotide repair patch pathway for processing of 8-oxoguanine in DNA.

Animals↗

PubMed1998

DNA repair and transcription in human premature aging disorders.

The human progeroid disorders Cockayne syndrome and Werner syndrome present with several clinical features that are associated with normal aging. These include distinct changes in the skin. The genes responsible for these conditions have recently been cloned and characterized. They both contain a characteristic helicase sequence, and helicase activity has been demonstrated using the purified Werner protein. Helicases are involved in a number of DNA metabolic transactions, including transcription, replication, and DNA repair. Cockayne cells are deficient in a special type of DNA repair, transcription coupled DNA repair, but they also appear to be defective in basal transcription. The diverse functions of the Cockayne protein are under intense study. Werner cells may have subtle defects in DNA repair, and possibly also in transcription. The biochemical clarification of the precise role of these gene products is likely to provide very significant clues into the mechanism of aging.

Aging↗

PubMed1997

Restoration of preferential and strand specific gene repair in group 2 Chinese hamster ovary mutants (UV5) by the XPD (ERCC2) gene.

It has recently been reported that the XPD (ERCC2) gene is an integral component of the basal transcription factor TFIIH. We have studied the direct role of this repair gene on the fine structure of DNA repair in hamster cells. The gene and strand specific DNA repair of UV induced pyrimidine dimers was determined in wild-type hamster cells, in hamster cells harboring a mutation in the gene homologous to the XPD gene and in mutant cells transfected with the human XPD gene. In the mutant cells, strand specific repair was severely deficient. In the transfected cells, preferential and strand specific gene repair were restored to wild-type levels. The results of the current study clearly demonstrate a direct role for the XPD gene product both in the preferential repair and bulk repair of pyrimidine dimers as well as its high functional conservation between rodent and human cells. An in vitro transcription assay was employed to investigate whether RNA polymerase II mediated transcription was also affected by the transfection with the XPD gene. No change in transcription between the mutant and transfected cells was observed. This suggests that the role of XPD in repair can be distinguished from its role in TFIIH dependent transcription initiation. Different functional domains of XPD appear to be necessary for repair versus transcription.

Animals↗

PubMed1995

Enzymes acting at strand interruptions in DNA.

Endogenous and environmental DNA-damaging agents often generate single-strand interruptions in DNA. The lesions trigger a complex set of cellular reactions. In most eukaryotic cells, cellular poly(ADP-ribose) formation is the most acute response to such damage. Recently, such events have been amenable to study with soluble cell-free extracts of human cells. These investigations clarify the modulating role on DNA repair by poly (ADP-ribose), and suggest that the primary function of this unusual polymer is to act as an antirecombinant agent. Similar biochemical studies of subsequent repair events have revealed a branched pathway for the ubiquitous DNA base excision-repair process. The alternative pathway provides the cell with back-up functions for individual steps in this essential form of DNA repair.

Animals↗

PubMed1994

Reconstitution of the DNA base excision-repair pathway.

BACKGROUND: The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes. RESULTS: We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I. CONCLUSIONS: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.

Bacterial Proteins↗

PubMed1994

Transcription and nucleotide excision repair--reflections, considerations and recent biochemical insights.

Recent years have witnessed considerable progress in the definition of the preferential repair of actively transcribed genes. Equally impressive progress has been achieved in our understanding of the genetic and biochemical complexity of the DNA-repair process called nucleotide excision repair (NER). Most recently studies in several laboratories have yielded observations which provide insights about how the processes of transcription and NER may be linked in prokaryotic and eukaryotic cells.

Animals↗

PubMed1994

Release of 5'-terminal deoxyribose-phosphate residues from incised abasic sites in DNA by the Escherichia coli RecJ protein.

Excision of deoxyribose-phosphate residues from enzymatically incised abasic sites in double-stranded DNA is required prior to gap-filling and ligation during DNA base excision-repair, and a candidate deoxyribophosphodiesterase (dRpase) activity has been identified in E. coli. This activity is shown here to be a function of the E. coli RecJ protein, previously described as a 5'-->3' single-strand specific DNA exonuclease involved in a recombination pathway and in mismatch repair. Highly purified preparations of dRpase contained 5'-->3' exonuclease activity for single-stranded DNA, and homogeneous RecJ protein purified from an overproducer strain had both 5'-->3' exonuclease and dRpase activity. Moreover, E. coli recJ strains were deficient in dRpase activity. The hydrolytic dRpase function of the RecJ protein requires Mg2+; in contrast, the activity of E. coli Fpg protein, that promotes the liberation of 5'-->3'Rp residues from DNA by beta-elimination, is suppressed by Mg2+. Several other E. coli nucleases, including exonucleases I, III, V, and VII, endonucleases I, III and IV and the 5'-->3' exonuclease function of DNA polymerase I, are unable to act as a dRpase. Nevertheless, E. coli fpg recJ double mutants retain capacity to repair abasic sites in DNA, indicating the presence of a back-up excision function.

Apurinic Acid↗

PubMed1992

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

Apurinic Acid↗

PubMed1991

Preferential recognition of I.T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase.

Double-stranded synthetic oligonucleotides with a centrally located dIMP residue in a 5'-32P-labeled strand were employed as substrates for hypoxanthine-DNA glycosylase. The enzyme activity was monitored by the generation of a piperidine-sensitive site in the labeled oligonucleotide. The enzyme was purified approximately 5000-fold from calf thymus. The purified enzyme removed efficiently a hypoxanthine base residue from an I.T base pair, but 15-20 times more slowly from an I.C base pair. Similar results were obtained with oligonucleotides in which the deoxyinosine residue was placed in different surrounding nucleotide sequences. The enzyme had no detectable activity on mismatched G.T, A.G or A.C base pairs. The data indicate that hypoxanthine-DNA glycosylase participates in the repair of deaminated adenine residues in DNA.

Base Composition↗