mutation dna

MUTATION RESEARCH-DNA REPAIR
vol. 3, issue 4, pp.201-348. 4 Maret 2004


Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy
Carine Chastel1, Josef Jiricny2 and Rolf Jaussi

Paul Scherrer Institut, CH-5232 PSI, Villigen, Switzerland

Received 29 August 2003; revised 25 November 2003; accepted 10 December 2003. ; Available online 4 January 2004.

Abstract
Radiotherapy is one of the principal modalities of cancer treatment, but the delivery of a curative dose of ionizing radiation (IR) to the tumour is frequently limited by the need to protect the normal tissues within the irradiated area from radiation damage. This problem could be circumvented if tumour cells could be selectively sensitized to killing by IR. One way to achieve this goal would be to transduce the tumour cells with expression vectors carrying toxin genes under the control of promoters that are inactive unless induced by IR. For this approach to be successful, two parameters must be met: (i) the expression vector has to be delivered to the tumour or its immediate vicinity (e.g. its vasculature) and (ii) the promoter driving the expression of the toxin gene has to have negligible basal activity, yet has to be activated by clinically-achievable doses of IR. Several vectors that fulfil these criteria are currently reaching clinical trials. In this review, we examine the response of mammalian cells to IR, and the current status of radiation-induced suicide gene therapy that is dependent on this response.
Author Keywords: Cancer; Immediate early genes; Ionizing radiation; p53; Promoter; Radiotherapy; X-rays













High frequency of nucleotide misincorporations upon the processing of double-strand breaks
Igor Kovalchuk, b and Olga Kovalchuka

a Department of Biological Sciences, University of Lethbridge, Lethbridge, Alta., Canada, T1K 3M4
b Friedrich Miescher-Institut, P.O. Box 2543, CH-4002, Basel, Switzerland

Accepted 30 September 2003. ; Available online 6 December 2003.

Abstract
Base substitutions were detected as a consequence of double-strand break (DSB) repair in plants. The fidelity of processing free DNA ends was analyzed using a stop-codon inactivated beta-glucuronidase (uidA) reporter gene. Circular and linear plasmids carrying the inactive gene were delivered to Nicotiana plumbaginifolia protoplasts or Nicotiana tabacum leaves. Processing of breaks which occurred in close proximity (5-9 bp) to termination codons led to occasional reversions and subsequent gene reactivation. In contrast, the repair of breaks occurring at a greater distance from the stop-codon resulted in a significantly lower number of reversions. The data suggest that the error prone processing of the free ends involves partial degradation and re-synthesis of the DNA repair substrate.
Author Keywords: DNA repair in plants; Strand break repair; Mutation frequency; Nucleotide misincorporation


















The kinase activity of DNA-PK is required to protect mammalian telomeres
Susan M. Baileya, d, 1, Mark A. Brennemanb, 1, JamesHalbrookc, Jac A. Nickoloffb, Robert L. Ullricha and Edwin H. Goodwin, d

a Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
b Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
c ICOS Corporation, Bothell, WA 98021, USA
d Biosciences Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545, USA

Received 11 June 2003; accepted 27 October 2003. ; Available online 4 December 2003.

Abstract
The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.
Author Keywords: DNA-PKcs; Mammalian telomeres; Double-strand breaks; DNA repair; Cancer
Abbreviations:DSB, double-strand break; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; TRF2, telomere repeat binding factor 2; FISH, fluorescence in situ hybridization; CO-FISH, chromosome-orientation fluorescence in situ hybridization; scid, severe combined immuno-deficiency; NHEJ, non-homologous end joining; HR, homologous recombination; BrdU, 5'-bromo-2'-deoxycytidine; BrdC, 5'-bromo-2'-deoxycytodine; DAPI, 4',6-diamidino-2-phenylindole
The isoflavonoids genistein and quercetin activate different stress
signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX
Ruiqiong Yea, Aaron A. Goodarzia, Ebba U. Kurza, Shin'ichi Saitob, Yuichiro Higashimotob, 1, Martin F. Lavinc, Ettore Appellab, Carl W. Andersond and Susan P. Lees-Miller a

a Department of Biochemistry & Molecular Biology, University of Calgary, 3330 Hospital Drive, N.W., Calgary, Alta., Canada T2N 4N1
b Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
c Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Brisbane 4029, Qld., Australia
d Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA

Accepted 27 October 2003. ; Available online 20 December 2003.

Abstract
The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets.
Author Keywords: p53; Genistein; Quercetin; Isoflavonoid; DNA damage; Phosphorylation
Abbreviations:A-T, ataxia telangiectasia; ATM, ataxia-telangiectasia mutated; BSA, bovine serum
albumin; DMSO, dimethyl sulphoxide; DNA-PK, DNA-dependent protein kinase; DSB, double-strand break; IR, ionizing radiation; NCS, neocarzinostatin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PIKK, phosphatidyl inositol 3 kinase-like protein kinase; SDS, sodium dodecyl sulphate; topo I, topoisomerase I; topo II, topoisomerase II; UV, ultraviolet radiation
AHNAK interacts with the DNA ligase IV–XRCC4 complex and stimulates DNA ligase IV-mediated double-stranded ligation
Thomas Stiff a, Emma Shtivelmanb, Penny Jeggo a and Boris Kyselaa

a Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK
b Cancer Research Institute, University of California at San Francisco, San Francisco, CA 94143, USA

Received 27 May 2003; revised 31 October 2003; accepted 6 November 2003. ; Available online 5 December 2003.

Abstract
AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV–XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV–XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction. This interaction is specific and is not observed with other DNA ligases nor with other components of the DNA non-homologous end-joining machinery. We characterised AHNAK as a protein that stimulates the double-stranded (DS) ligation activity of DNA ligase IV–XRCC4. We show that AHNAK has weak DNA-binding activity and forms a stable complex with the DNA ligase IV–XRCC4 complex on DNA. AHNAK is also able to link two DNA molecules to a similar extent to that previously reported for Ku. Together, these findings demonstrate new activities for AHNAK, and raise the possibility that it may function to modulate DNA non-homologous end-joining.
Author Keywords: DNA non-homologous end-joining; Double-stranded ligation; AHNAK; Neuroblastoma cell lines








New immunoaffinity-LC-MS/MS methodology reveals that Aag null mice are deficient in their ability to clear 1,N6-etheno-deoxyadenosine DNA lesions from lung and liver in vivo
Amy-Joan L. Hama, b, Bevin P. Engelwarde, Hasan Kocc, d, Ramiah Sangaiahc, Lisiane B. Meirae, Leona D. Samson, , e and James A. Swenberga, c

a Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
b Department of Biochemistry and Proteomics Laboratory, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
c Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC, USA
d Present address: Huck Institute for Life Sciences, Pennsylvania State University, University Park, PA 16802, USA
e Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Received 12 August 2003;  accepted 6 November 2003. ; Available online 7 December 2003.

Abstract
The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine (dA). Previous attempts to determine the in vivo role of Aag in dA repair were complicated by technological difficulties in measuring low levels of dA in genomic DNA. Here we describe the development of a new method for dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal dA bases, these lesions can also be eliminated from the genome via an alternative mechanism.
Author Keywords: Immunoaffinity-LC-MS/MS methodology; 1,N6-ethenodeoxyadenosine; Lung; Liver; Alkyladenine DNA glycosylase; Mice





Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system
Kyung-Jong Leea, b, 1, Marko Jovanovica, Durga Udayakumara, Catherine L. Bladena and William S. Dynana

a Institute of Molecular Medicine and Genetics, Medical College of Georgia, Room CB-2803, Augusta, GA 30912, USA
b Department of Biochemistry and Molecular Biology, Medical College of Georgia, Room CB-2803, Augusta, GA 30912, USA

Accepted 7 November 2003. ; Available online 12 December 2003.

Abstract
Nonhomologous end joining (NHEJ) is the principal mechanism for repairing DNA double-strand breaks in mammalian cells. NHEJ requires at least three protein components: the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku protein, and the DNA ligase IV/XRCC4 (DNL IV/XRCC4) complex. Although DNA-PKcs phosphorylates several sites within itself and these other proteins, the significance of phosphorylation at individual sites is not yet understood. Here we investigate the effects of DNA-PKcs-mediated phosphorylation at two sites in XRCC4. One is a previously described site at serine 260; the other is a newly mapped site at serine 318. XRCC4 bearing mutations at these sites was co-expressed with DNL IV, the resulting complexes were purified, and activity was tested in a cell-free end-joining system reconstituted from recombinant and purified proteins. Substitution of alanine for serine 260 or 318, which prevents phosphorylation at these positions, or aspartate for serine 260, which mimics constitutive phosphorylation, had no significant effect on overall end-joining activity. In the assay system used, DNA-PKcs is not essential, but when present, arrests the reaction until phosphorylation occurs, in effect establishing a reaction checkpoint. Mutations at serines 260 and 318 did not affect establishment or release from the checkpoint. Results demonstrate that DNA-PKcs-mediated phosphorylation of XRCC4 serine 260 and serine 318 does not directly control end-joining under the conditions tested.

Author Keywords: XRCC4; DNA-PKcs; Phosphorylation; NHEJ; Double-strand breaks
Abbreviations:NHEJ, nonhomologous end joining; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DNL IV, DNA ligase IV; GST-X4, glutathione-S-transferase-XRCC4







The yeast Rad7/Rad16/Abf1 complex generates superhelical torsion in DNA that is required for nucleotide excision repair
Shirong Yua, Tom Owen-Hughesb, Errol C. Friedbergc, Raymond Watersa and Simon H. Reed a, c

a Department of Pathology, University of Wales College of Medicine, Cardiff CF14 4XN, Wales, UK
b Division of Gene Regulation, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, UK
c Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA

Received 12 August 2003; accepted 11 November 2003. ; Available online 12 December 2003.

Abstract
Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER––a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA.

Author Keywords: Nucleotide excision repair; Saccharomyces cerevisiae; Abf1; Rad7; Rad16; Supercoiling





8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site
Martine E. Lomax, , Siobhan Cunniffe and Peter O'Neill

Radiation and Genome Stability Unit, Medical Research Council, Harwell, Didcot, Oxfordshire OX11 0RD, UK

Received 10 September 2003;  revised 19 November 2003;  accepted 19 November 2003. ; Available online 12 December 2003.

Abstract
Ionising radiation produces clustered DNA damage. Recent studies have established that the efficiency of excision of a lesion within clustered damage sites is reduced. This study presents evidence that the repair of clustered DNA damage is compromised, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to eight fold. Simple clustered damage sites, comprised of a single-strand break, one or five bases 3' or 5' to 8-oxoG on the opposite strand, were synthesised in oligonucleotides and repair carried out in XRS5 nuclear extracts. The rate of repair of the single-strand break within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase III/XRCC1 complex. The single-strand break, present as an isolated lesion, is repaired by short-patch base excision repair, however the mechanism of repair of the single-strand break within the clustered damage site is asymmetric. When the lesions are 5' to each other, the single-strand break is rejoined by short-patch repair whereas the rejoining of the single-strand break occurs by long-patch type repair when the lesions are 3' to one another. The retardation of DNA ligase III/XRCC1 complex, following addition of one base, is responsible for the initiation of long-patch base excision repair when the lesions are 3' to each other. The lesions within the cluster are processed sequentially, the single-strand break being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage is suggested to have implications for mutation induction by radiation.
Author Keywords: Clustered DNA damage; Single-strand breaks; 8-OxoG; Base excision repair




Identification of specific amino acid residues in the E. coli  processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'
Jill M. Duzena, Graham C. Walkerb and Mark D. Sutton, , a

a Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214, USA
b Biology Department, 68-633 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

Received 17 September 2003;  revised 18 November 2003;  accepted 20 November 2003. ; Available online 20 December 2003.

Abstract
Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli  sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each  protomer. Although both UmuC (DNA pol V) and the  catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD' do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on  important for its interactions with the two forms of the umuD gene product. We also characterized the surface of  important for its interaction with the  catalytic subunit of pol III. Our results indicate that although UmuD, UmuD' and  share some common contacts with , each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD' with  impose a DNA damage-responsive conditionality on how  interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases.
Author Keywords: DNA replication; Translesion DNA synthesis; DNA polymerase management; umuDC;  sliding clamp; dnaN



Stereoselective excision of thymine glycol lesions by mammalian cell extracts
Monica M. McTigue, Robert A. Rieger, Thomas A. Rosenquist, Charles R. Iden and Carlos R. de los Santos,

Department of Pharmacological Sciences, State University of New York, Stony Brook, NY 11794-8651, USA

Accepted 20 November 2003. ; Available online 19 December 2003.

Abstract
Thymine glycols (Tg) are major pyrimidine oxidation products produced by chemical agents and ionizing radiation. Recent improvements in purification procedures gave us the opportunity to examine the incision of DNA duplexes containing a single (5S,6R)- or (5R,6S)-Tg lesion by mouse NTH1 DNA glycosylase and mammalian cell nuclear extracts. Time course experiments and steady state enzyme kinetics indicated that mNTH1 discriminates between the cis-Tg isomers. In addition, a variety of mammalian cell nuclear extracts showed a similar discrimination between the cis-Tg isomers. Trapping of Schiff base intermediates with sodium borohydride demonstrated that a single protein–DNA complex was formed in the presence of the nuclear extracts. The electrophoretic mobility of trapped complexes formed with both Tg isomers was identical to one another and similar to that of the complex formed with recombinant mNTH1. These results suggest that among all Tg-active DNA glycosylases, NTH1 is the major enzyme in mammalian cell nuclear extracts responsible for incision of duplexes containing cis-Tg isomers.
Author Keywords: DNA damage; Thymine glycol isomers; NTH DNA glycosylase
Abbreviations:HPLC, high-pressure liquid chromatography; GC/MS, gas chromatography/mass spectroscopy; LC/MS, liquid chromatography/mass spectroscopy; LC/MS/MS, liquid chromatography/tandem mass spectroscopy; PAGE, polyacrylamide gel electrophoresis; Tg, thymine glycol; NEIL1, Nei-like 1 DNA glycosylase; NEIL2, Nei-like 2 DNA glycosylase; TGG1, thymine glycol DNA glycosylase 1; TGG2, thymine glycol DNA glycosylase 2


A role for DNA polymerase V in GT mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TT sequence in E. coli
Jun Yin1, Kwang Young Seo1 and Edward L. Loechler,

Biology Department, Boston University, Boston, MA 02215, USA

Received 8 October 2003;  revised 25 November 2003;  accepted 25 November 2003. ; Available online 8 January 2004.

Abstract
Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GCTA, GCAT, etc.) principally via its major adduct [+ta]-B[a]P-N2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P-N2-dG built into a plasmid in a 5'-TT sequence gives approximately equal numbers of GT and GA mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in GT versus GA mutations. GT mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V (umuD/C), demonstrating a role for damage-inducible DNA Pol V in a GT pathway. GT mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5'-GA sequence, and showed that the frequency of GT mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two GT mutagenic pathways exist for [+ta]-B[a]P-N2-dG, where sequence context dictates which pathway is followed. In contrast to the GT mutations, herein GA mutations from [+ta]-B[a]P-N2-dG in the 5'-TT sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that GA mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P-N2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two GA mutagenic pathways for [+ta]-B[a]P-N2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both GT and GA mutations from [+ta]-B[a]P-N2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.
Author Keywords: Cancer; Genotoxins; Mutations; Mutational spectrum; Adduct site-specific mutagenesis; Lesion and sequence context effects; Pol II; Pol IV; Pol V; UVM
Abbreviations:B[a]P, benzo[a]pyrene; (+)-anti-B[a]PDE, 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; [+ta]-B[a]P-N2-dG, the major adduct of (+)-anti-B[a]PDE, formed by trans addition of N2-dG to (+)-anti-B[a]PDE (see Fig. 1); PAH, polycyclic aromatic hydrocarbon; AAF-C8-dG, the major adduct of N-2-acetylaminofluorene, formed by trans addition of C8-dG to N-2-acetylaminofluorene; TT-CPD, cis-syn-TT cyclopyrimidine dimer, a UV photoproduct; CC-CPD, cis-syn-CC cyclopyrimidine dimer, a UV photoproduct; T(6-4)T, the UV photoproduct formed via a covalent bond between the 5'-C6-dT and 3'-C4-dT of adjacent thymines, followed by additional chemical rearrangements transforming the 3'-dT into a pyrimidone

Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae
Daisuke Maeda, Masayuki Seki, , Fumitoshi Onoda, Dana Branzei, Yoh-ichi Kawabe and Takemi Enomoto

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan

Accepted 27 November 2003. ; Available online 30 December 2003.

Abstract
Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV.
Author Keywords: Ubc9; Rad6; Rad52; SUMO; Recombination; Sgs1

An integrated mechanistic model for transcription-coupled nucleotide excision repair*1
Shwetal Patel, , a, 1, K. V. Venkateshb and Jeremy S. Edwardsa

a Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA
b Department of Chemical Engineering, Indian Institute of Technology, Bombay, Powai, Mumbai-400076, India

Received 9 October 2003;  accepted 21 November 2003. ; Available online 29 December 2003.

Abstract
Preferential repair of the transcribed strand of active genes is usually attributed to a coupling protein that dislodges RNA polymerase stalled at a damage site and recruits repair enzymes. Experimental observations of the effect of transcription on preferential repair in Escherichia coli are contradictory and inexplicable by this model. In this study, it is argued that the multiple conformations displayed by a stalled RNA polymerase result in two sub-pathways for repair: Mfd coupled and direct. Together with the fact that RNA polymerase recruits the repair enzymes in a promoter dependent manner, an integrated mechanistic model is proposed that is capable of explaining the effect of transcription on preferential repair reported in literature. The quantitative behavior of the model is illustrated by describing the various reactions using a biochemical network. The implications of the model on the mechanism for transcription-coupled repair in higher organisms are briefly discussed.
Author Keywords: Cockayne syndrome; In silico biology; Hypothesis; UvrABCD