Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts.

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.

Two forms of UvrC protein with different double-stranded DNA binding affinities.

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.

bicaudal encodes the Drosophila beta NAC homolog, a component of the ribosomal translational machinery*.

bicaudal was the first Drosophila mutation identified as producing mirror-image pattern duplications along the anteroposterior axis of the embryo. However the mutation has been little studied due to its low penetrance and suppressibility. We undertook cloning of the bicaudal locus together with studies of the mutation's effects on key elements of the posterior embryonic patterning pathway. Our mapping studies place the bicaudal mutation within a approximately 2 kb region, 3' to the protein coding sequence of the Drosophila homolog of beta NAC, a subunit of Nascent polypeptide Associated Complex (NAC). Genomic DNA encoding beta NAC completely rescues the bicaudal phenotype. The lethal phenotype of Enhancer of Bicaudal, E(Bic), a mutation hypothesized to affect the bicaudal locus, is also completely rescued by the beta NAC locus. We further demonstrate that the E(Bic) mutation is caused by a P element insertion into the transcribed region of the beta NAC gene. NAC is among the first ribosome-associated entities to bind the nascent polypeptide after peptide bond formation. In contrast to other bicaudal-embryo-producing mutations, bicaudal leads to ectopic translation of mRNA for the posterior determinant nanos, without affecting the localization of mRNA for its upstream regulator, oskar, in the embryo. These findings suggest that repression of nanos mRNA translation occurs on the ribosome and involves a role for beta NAC.

Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis.

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.

A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair.

The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.

Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products.

We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.

3B55: a repetitious sequence family which is transcribed and proportionately replicated in germ-line polyploid nuclei of Calliphora erythrocephala.

The chromosomes of dipteran polyploid nurse cell nuclei are functionally analogous to the oocyte lampbrush chromosomes of the Amphibia. In investigating the transcriptional and replicative activity of these nuclei in Calliphora erythrocephala we have identified a cloned highly repetitious DNA fragment which shows enhanced transcriptional activity in these cells and different replicative behavior in these germ-line polyploid nuclei as opposed to somatic polytene nuclei. The clone, 3B55, contains a 6.8-kb insert which consists primarily of tandemly repeated 200-bp sequences defined by RsaI sites. From Southern hybridizations to diploid (embryonic) genomic DNA, 3B55-related DNA was calculated to represent a significant fraction of the haploid genome (0.8% or 5000 kb). In situ hybridizations established that these sequences are present in the pericentric regions of four of the six chromosomes. Thus the 3B55 sequences have the properties of a satellite-type DNA family. Quantitation of the 3B55 200-bp monomer (which represents approximately 60% of the genomic 3B55 DNA sequences in all tissues examined) revealed that in somatic polytene salivary gland nuclei, 3B55 DNA is highly under-replicated to give a monomer representation of only 48 +/- 11 kb per haploid genome. However, in germ-line nurse cell nuclei, 3B55 DNA is proportionately replicated to give a monomer genomic representation (3560 +/- 344 kb) equivalent to that of diploid DNA (3011 +/- 202 kb). Transcripts complementary to 3B55 sequences are at least 25 times more abundant in total nurse cell nuclear RNA than in total embryonic nuclear RNA. These findings suggest an association of the 3B55 sequence family with some germ-line specific function.

Effects of cis-diamminedichloroplatinum (CDDP) on HeLa cell non-histone nuclear proteins.

Two-dimensional isoelectric focusing and sodium dodecyl sulfate (SDS) gel electrophoretic analyses of successive salt extracts of purified HeLa cell nuclei were used to study the effects of cis-diamminedichloroplatinum (CDDP) (cisplatin) on the synthesis and extractability of nuclear non-histone proteins. Nuclei were extracted sequentially with 0.025 M NaCl-0.075 M EDTA, 0.01 M Tris, and 0.6 M NaCl. Each fraction contained 100-400 polypeptide spots, only a few of which were affected by a 3.5-h CDDP pretreatment of the cells. The biosynthesis and/or metabolism of four polypeptide spots was significantly affected by the CDDP treatment. These polypeptides included: (a) 36K/5.8 (designated by MW/PI) in the 0.025 M NaCl-0.075 M EDTA extract, which decreased in intensity with treatment at 10 micrograms CDDP/ml; (b) polypeptide spots 50K/6.0 and 45K/5.3 in the Tris extract, which increased in intensity over a range of CDDP concentrations of 0-5 microgram CDDP/ml; and (c) a polypeptide complex at 110K/7.7 in the 0.6 M Na Cl extract, which decreased in intensity at CDDP concentrations of 0-5 microgram CDDP/ml. Scanning densitometry of the protein spots of the 0.6 M NaCl extract demonstrated that the 110K/7.7 complex decreased to half its intensity compared with non-drug-treated controls at a CDDP concentration of 0.9 microgram CDDP/ml. We have found that high-resolution two-dimensional gel electrophoretic analysis of nuclear proteins is a valuable technique for studying the effects of cytotoxic agents on the synthesis and/or extractability of specific cellular proteins.