Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria.

DNA repair alkyltransferases protect organisms against the cytotoxic, mutagenic, and carcinogenic effects of alkylating agents by transferring alkyl adducts from DNA to an active cysteine on the protein, thereby restoring the native DNA structure. We used random sequence substitutions to gain structure-function information about the human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63), as well as to create active mutants. Twelve codons surrounding but not including the active cysteine were replaced by a random nucleotide sequence, and the resulting random library was selected for the ability to provide alkyltransferase-deficient Escherichia coli with resistance to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Few amino acid changes were tolerated in this evolutionarily conserved region of the protein. One mutation, a valine to phenylalanine change at codon 139 (V139F), was found in 70% of the selected mutants; in fact, this mutant was selected much more frequently than the wild type. V139F provided alkyltransferase-deficient bacteria with greater protection than the wild-type protein against both the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine, increasing the D37 over 4-fold and reducing the mutagenesis rate 2.7-5.5-fold. This mutant human alkyltransferase, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of alkylation-based chemotherapeutic regimens.

Compositional differences within and between eukaryotic genomes

Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is ρ*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size ≥50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection {ρ*XY} as the genome signature. This paper identifies ρ*XY extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.

Association of terminal deoxynucleotidyl transferase with Ku

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein–protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

The BRCA2 gene product functionally interacts with p53 and RAD51

Germ-line mutations in the human BRCA2 gene confer susceptibility to breast cancer. Efforts to elucidate its function have revealed a putative transcriptional activation domain and in vitro interaction with the DNA repair protein RAD51. Other studies have indicated that RAD51 physically associates with the p53 tumor suppressor protein. Here we show that the BRCA2 gene product is a 460-kDa nuclear phosphoprotein, which forms in vivo complexes with both p53 and RAD51. Moreover, exogenous BRCA2 expression in cancer cells inhibits p53’s transcriptional activity, and RAD51 coexpression enhances BRCA2’s inhibitory effects. These findings demonstrate that BRCA2 physically and functionally interacts with two key components of cell cycle control and DNA repair pathways. Thus, BRCA2 likely participates with p53 and RAD51 in maintaining genome integrity.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

Yeast and human genes that affect the Escherichia coli SOS response

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the Escherichia coli SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g., RAD51 and HHT1 as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.

The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of α-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of α-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The α-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of αAmy3 3′ untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire αAmy3 3′ UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire αAmy3 3′ UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the αAmy3 3′ UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the α-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Highly efficient base excision repair (BER) in human and rat male germ cells

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.

Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination

Alternative reproductive cycles make use of different strategies to generate different reproductive products. In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR. The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several rec gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Regulation of SOS mutagenesis by proteolysis.

DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of the UmuD (UmuD') and UmuC proteins. The intracellular level of these proteins is tightly regulated at both the transcriptional and the posttranslational levels. Such regulation presumably allows cells to deal with DNA damage via error-free repair pathways before being committed to error-prone pathways. We have recently discovered that as part of this elaborate regulation, both the UmuD and the UmuC proteins are rapidly degraded in vivo. We report here that the enzyme responsible for their degradation is the ATP-dependent serine protease, Lon. In contrast, UmuD' (the posttranslational product and mutagenically active form of UmuD) is degraded at a much reduced rate by Lon, but is instead rapidly degraded by another ATP-dependent protease, ClpXP. Interestingly, UmuD' is rapidly degraded by ClpXP only when it is in a heterodimeric complex with UmuD. Formation of UmuD/UmuD' heterodimers in preference to UmuD' homodimers therefore targets UmuD' protein for proteolysis. Such a mechanism allows cells to reduce the intracellular levels of the mutagenically active Umu proteins and thereby return to a resting state once error-prone DNA repair has occurred. The apparent half-life of the heterodimeric UmuD/D' complex is greatly increased in the clpX::Kan and clpP::Kan strains and these strains are correspondingly rendered virtually UV non-mutable. We believe that these phenotypes are consistent with the suggestion that while the UmuD/D' heterodimer is mutagenically inactive, it still retains the ability to interact with UmuC, and thereby precludes the formation of the mutagenically active UmuD'2C complex.

Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase.

Agents that damage DNA in Escherichia coli or interfere with its replication induce DNA repair and mutagenesis via the SOS response. This well-known activity is regulated by the RecA protein and the LexA repressor. Following repair or bypass of the DNA lesion, the cell returns to its resting state by a largely unknown process. We found that 2-keto-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase; EC 4.1.3.16) is necessary for the recovery of respiration and that it is regulated by the SOS response. This protein was induced by DNA-damaging agents. Induction required RecA activation. When the LexA regulon was repressed, activation of RecA was not sufficient for induction, indicating the requirement for an additional protein under LexA control. Finally, a mutant in the corresponding hga gene was UV sensitive. 2-Keto-4-hydroxyglutarate aldolase also plays a role in respiratory metabolic pathways, which suggests a mechanism for respiration resumption during the termination of the SOS response.