Biomedical subjects

A K McCullough

Publications and source records attributed to A K McCullough.

9 recordsLinked to original sources

PubMed2001

hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs.

The human MutY homolog, hMYH, is an adenine-specific DNA glycosylase that removes adenines or 2-hydroxyadenines mispaired with guanines or 8-oxoguanines. In order to prevent mutations, this activity must be directed to the newly synthesized strand and not the template strand during DNA synthesis. The subcellular localization and expression of hMYH has been studied in serum-stimulated, proliferating MRC5 cells. Using specific antibodies, we demonstrate that endogenous hMYH protein localized both to nuclei and mitochondria. hMYH in the nuclei is distinctly distributed and co-localized with BrdU at replication foci and with proliferating cell nuclear antigen (PCNA). The levels of hMYH in the nucleus increased 3- to 4-fold during progression of the cell cycle and reached maximum levels in S phase compared to early G(1). Similar results were obtained for PCNA, while there were no notable changes in expression of 8-oxoguanine glycosylase or the human MutT homolog, MTH1, throughout the cell cycle. The cell cycle-dependent expression and localization of hMYH at sites of DNA replication suggest a role for this glycosylase in immediate post-replication DNA base excision repair.

Adenine↗

PubMed2001

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites.

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.

Bacteriophage T4↗

PubMed2000

AP lyases and dRPases: commonality of mechanism.

Enzymes that release 5'-deoxyribose-5-phosphate (dRP) residues from preincised apurinic/apyrimidinic (AP) DNA have been collectively termed DNA deoxyribophosphodiesterases (dRPases), but they fall into two distinct categories: the hydrolytic dRPases and AP lyases. In order to resolve a number of conflicting reports in the dRPase literature, we examined two putative hydrolytic dRPases (Escherichia coli exonuclease I (exo I) and RecJ) and four AP lyases (E. coli 2, 6-dihydroxy-5N-formamidopyrimidine (Fapy) DNA glycosylase (Fpg) and endonuclease III (endo III), bacteriophage T4 endonuclease V (endo V), and rat polymerase beta (beta-pol)) for their abilities to (i) excise dRP from preincised AP DNA and (ii) incise AP DNA. Although exo I and RecJ exhibited robust 3' to 5' and 5' to 3' exonucleolytic activities, respectively, on appropriate substrates, they failed to demonstrate detectable dRPase activity. All four AP lyases possessed both dRPase and traditional AP lyase activities, albeit to varying degrees. Moreover, as best illustrated with Fpg, AP lyase enzymes could be trapped on both preincised and unincised AP DNA using NaBH(4) as the reducing agent. These results further support the assertion that the catalytic mechanism of the AP lyases, the beta-elimination reaction, does proceed through an imine enzyme-DNA intermediate and that the active site residues responsible for dRP release must contain primary amines. Further, these data indicate a biological significance for the beta-elimination reaction of DNA glycosylase/AP lyases in that they, in concert with hydrolytic AP endonucleases, can create appropriate gapped substrates for short patch base excision repair (BER) synthesis to occur efficiently.

Animals↗

PubMed2000

Intron conservation in a UV-specific DNA repair gene encoded by chlorella viruses.

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5'-AG/GTATGT and 3'-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences.

Amino Acid Sequence↗

PubMed1999

Initiation of base excision repair: glycosylase mechanisms and structures.

The base excision repair pathway is an organism's primary defense against mutations induced by oxidative, alkylating, and other DNA-damaging agents. This pathway is initiated by DNA glycosylases that excise the damaged base by cleavage of the glycosidic bond between the base and the DNA sugar-phosphate backbone. A subset of glycosylases has an associated apurinic/apyrimidinic (AP) lyase activity that further processes the AP site to generate cleavage of the DNA phosphate backbone. Chemical mechanisms that are supported by biochemical and structural data have been proposed for several glycosylases and glycosylase/AP lyases. This review focuses on the chemical mechanisms of catalysis in the context of recent structural information, with emphasis on the catalytic residues and the active site conformations of several cocrystal structures of glycosylases with their substrate DNAs. Common structural motifs for DNA binding and damage specificity as well as conservation of acidic residues and amino groups for catalysis are discussed.

Catalysis↗

PubMed1998

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1.

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

Binding Sites↗

PubMed1997

The role of base flipping in damage recognition and catalysis by T4 endonuclease V.

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5'-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3'-thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.

2-Aminopurine↗

PubMed1997

Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV.

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.

Amino Acid Sequence↗

PubMed1996

Structural determinants for specific recognition by T4 endonuclease V.

DNA glycosylases catalyze the scission of the N-glycosyl bond linking either a damaged or mismatched base to the DNA sugar phosphate backbone. T4 endonuclease V is a glycosylase/apurinic (AP) lyase that is specific for UV light-induced cis-syn pyrimidine dimers. As a proposed transition state analog/inhibitor for glycosylases, a phosphoramidite derivative containing a pyrrolidine residue has been synthesized. The binding of endonuclease V to this duplex was analyzed by gel mobility shift assays and resulted in a single stable complex of reduced mobility and an apparent Kd of 17 nM. To assess the importance of the positive charge for specific binding, studies using other non-cleavable substrate analogs were performed. Wild type T4 endonuclease V shows an 8-fold decreased affinity for a tetrahydrofuran as compared with the pyrrolidine residue, demonstrating the significance of the positive charge for recognition. A 2-fold increase in binding affinity for a reduced AP site was observed. Similar assays using catalytically compromised mutants (E23Q and E23D) of endonuclease V demonstrate altered affinities for the pyrrolidine as well as tetrahydrofuran and reduced AP sites. This approach has provided insight into the structural mechanism by which specific lesions are targeted by the protein as well as the structural determinants of the DNA required for specific recognition by T4 endonuclease V.

DNA Repair↗