Grigory L. Dianov

Human DNA polymerase β initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short‐patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase β (Pol β) is responsible for the repair synthesis in this pathway and also removes a 5′‐sugar phosphate residue by catalyzing a β‐elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to β‐elimination are removed through a long‐patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol β, Pol δ and Pol ϵ) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to β‐elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol β is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long‐patch base excision repair synthesis.

Base excision repair in nuclear and mitochondrial DNA.

Base excision repair mechanisms have been analyzed in nuclear and mitochondrial DNA. We measured the size and position of the newly incorporated DNA repair patch in various DNA substrates containing single oxidative lesions. Repair of 8-oxoguanine and of thymine glycol is almost exclusively via the base excision repair (BER) pathway with little or no involvement of nucleotide excision repair (NER). The repair mode is generally via the single-nucleotide replacement pathway with little incorporation into longer patches. Extension of these studies suggests that DNA polymerase beta plays a critical role not only in the short-patch repair process but also in the long-patch, PCNA-dependent pathway. Mitochondria are targets for a heavy load of oxidative DNA damage. They have efficient BER repair capacity, but cannot repair most bulky lesions normally repaired by NER. In vitro experiments performed using rat and human mitochondrial extracts suggest that the repair incorporation during the removal of uracil in DNA occurs via the short-patch repair BER pathway. Oxidative DNA damage accumulates with age in mitochondrial DNA, but this cannot be explained by an attenuation of DNA repair. In contrast, we observe that mitochondrial incision of 8-oxoG increases with age in rodents.

An Oxidative Damage-specific Endonuclease from Rat Liver Mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE’s molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mm KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mm EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE’s activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.

Oxidative DNA damage processing in nuclear and mitochondrial DNA.

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, to examine the repair of different types of oxidative 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.

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.

DNA Repair and Transcription in Premature Aging Syndromes

The human progeroid disorders Cockayne syndrome (CS) and Werner syndrome (WS) exhibit several clinical features that are associated with normal aging. With the recent cloning of the Werner syndrome (WRN) gene, and with the information that this gene, the CS complementation group B (CS-B) gene, and some XP gene products are putative helicases and involved in nucleic acid metabolism, further understanding the molecular deficiency in these disorders is a high priority. Helicases are involved in a number of DNA metabolic activities including transcription, replication and DNA repair. These human disorders provide excellent model systems for studies on aging. The patients have many signs and symptoms of normal aging, but they are segmental progeroid diseases, indicating that some features of normal aging are not seen. The function of the CS-B and WRN proteins appear to be at the crossroads of aging, DNA repair, DNA replication, and transcription, and hence these studies nicely combine our mechanistic interest in basic processes with our interest in aging. CS (Group B), WRN, and other age-related disorders (Bloom, Xeroderma pigmentosum Groups B and D) carry mutations in related genes characterized by conserved motifs of sequence homology. Some proteins of this family have been demonstrated to be DNA-dependent ATPases, a subset of which have also been shown to be helicases. Proteins of this family are involved in various aspects of chromosome metabolism. The molecular defects responsible for the clinical phenotypes of these diseases remain to be determined, but presumably relate to the functional activities of these conserved proteins. In addition, specific protein-DNA and protein-protein interactions are likely to play critical roles in cellular function. Cells derived from CS patients 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 CSB protein are under intense study. Werner syndrome cells may have subtle defects in DNA repair, and possibly also in transcription. The biochemical clarification of the precise role(s) of these gene products is likely to provide very significant clues into the mechanism of aging.