Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro.

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle.

Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=(m)CG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs.

Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis.

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B.

Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate.

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase η opposite the T that results from deamination of the methylC ((m)C) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of (m)C deamination in a CPD has not been previously studied.

Methyl CpG binding protein 2 (MeCP2) enhances photodimer formation at methyl-CpG sites but suppresses dimer deamination.

Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of C→T mutations, but occurs with a half life of ∼50 000 years. In contrast, cytosine within sunlight induced cyclobutane dipyrimidine dimers (CPD's), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlate with C→T mutation hotspots in skin cancers.

Acceleration of 5-methylcytosine deamination in cyclobutane dimers by G and its implications for UV-induced C-to-T mutation hotspots.

Sunlight-induced C-->T mutation hotspots occur most frequently at methylated CpG sites in tumor suppressor genes and are thought to arise from translesion synthesis past deaminated cyclobutane pyrimidine dimers (CPDs). While it is known that methylation enhances CPD formation in sunlight, little is known about the effect of methylation and sequence context on the deamination of 5-methylcytosine ((m)C) and its contribution to mutagenesis at these hotspots. Using an enzymatic method, we have determined the yields and deamination rates of C and (m)C in CPDs and find that the frequency of UVB-induced CPDs correlates with the oxidation potential of the flanking bases.

Ability of polymerase eta and T7 DNA polymerase to bypass bulge structures.

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol eta with primer-templates containing bulge structures by single round primer extension.

DNA synthesis past a 5-methylC-containing cis-syn-cyclobutane pyrimidine dimer by yeast pol eta is highly nonmutagenic.

Cyclobutane pyrimidine dimers (CPDs) are responsible for a considerable fraction of sunlight-induced C to T and 5-methycytosine (mC) to T mutations in mammalian cells, though the precise mechanism is unknown. One possibility is that the C or mC of a CPD is not mutagenic and must first deaminate to U or T, respectively, for A to be inserted by a DNA polymerase. Alternatively, A might be directly inserted opposite the C or mC prior to deamination via an E-imino tautomer of the C or mC or by a nontemplated mechanism in which the photoproduct is sterically excluded from the active site.

DNA-thumb interactions and processivity of T7 DNA polymerase in comparison to yeast polymerase eta.

The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3'-end of the primer-template, but does not show how thioredoxin confers processivity to the polymerase. In the crystal structure highly conserved amino acids Asn(335) and Ser(338) of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand.

Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch.

In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P to form CheY and Pi. This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch. In particular we have identified the phosphatase as FliY, which resembles E.