John-Stephen Taylor

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis.

The expression of the Escherichia coli DNA polymerases pol V (UmuD′ 2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6–4) photoproducts, TT cis–syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol IIIs processivity β,γ-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic ‘signature’ for pol V is its incorporation of guanine opposite the 3′-thymine of a TT (6–4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10-3 to 10-4, with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and β,γ-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require β,γ-complex in the presence of non-hydrolysable ATPγS, indicating that an intact RecA filament may be required for translesion synthesis.

Crystal structure of a DNA decamer containing a cis-syn thymine dimer

It is well known that exposure to UV induces DNA damage, which is the first step in mutagenesis and a major cause of skin cancer. Among a variety of photoproducts, cyclobutane-type pyrimidine photodimers (CPD) are the most abundant primary lesion. Despite its biological importance, the precise relationship between the structure and properties of DNA containing CPD has remained to be elucidated. Here, we report the free (unbound) crystal structure of duplex DNA containing a CPD lesion at a resolution of 2.0 Å. Our crystal structure shows that the overall helical axis bends ≈30° toward the major groove and unwinds ≈9°, in remarkable agreement with some previous theoretical and experimental studies. There are also significant differences in local structure compared with standard B-DNA, including pinching of the minor groove at the 3′ side of the CPD lesion, a severe change of the base pair parameter in the 5′ side, and serious widening of both minor and major groves both 3′ and 5′ of the CPD. Overall, the structure of the damaged DNA differs from undamaged DNA to an extent that DNA repair proteins may recognize this conformation, and the various components of the replicational and transcriptional machinery may be interfered with due to the perturbed local and global structure.

Shape effects of nanoparticles conjugated with cell-penetrating peptides (HIV Tat PTD) on CHO cell uptake

In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective cross-linking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese hamster ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.

Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

SUMMARY Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.

Reaction Mechanism of (6-4) Photolyase

The (6-4) photolyase catalyzes the photoreversal of the (6-4) dipyrimidine photoproducts induced in DNA by ultraviolet light. Using the cloned Drosophila melanogaster (6-4) photolyase gene, we overproduced and purified the recombinant enzyme. The binding and catalytic properties of the enzyme were investigated using natural substrates, T[6-4]T and T[6-4]C, and the Dewar isomer of (6-4) photoproduct and substrate analogs s5T[6-4]T/thietane, mes5T[6-4]T, and theN-methyl-3′T thietane analog of the oxetane intermediate. The enzyme binds to the natural substrates and to mes5T[6-4]T with high affinity (K D ∼10−9-10−10 m) and produces a DNase I footprint of about 20 base pairs around the photolesion. Several lines of evidence suggest that upon binding by the enzyme, the photoproduct flips out of the duplex. Of the four substrates that bind with high affinity to the enzyme, T[6-4]T and T[6-4]C are repaired with relatively high quantum yields compared with the Dewar isomer and the mes5T[6-4]T which are repaired with 300–400-fold lower quantum yield than the former two photoproducts. Reduction of the FAD cofactor with dithionite increases the quantum yield of repair. Taken together, the data are consistent with photoinduced electron transfer from reduced FAD to substrate, in a manner analogous to the cyclobutane pyrimidine dimer photolyase.

New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions.

The A-rule in mutagenesis was originally proposed to explain the preponderance of X-->T mutations observed for abasic sites and UV damaged sites. It was deduced that when a polymerase was faced with a non-instructional lesion, typified by an abasic site, it would preferentially incorporate an A. In the absence of any other compelling explanation, any lesion causing an X-->T mutation has often been classified as non-instructional to account for its apparent lack of instructional ability. The A-rule and the classification of lesions as non-instructional were formulated before the active sites of any polymerases or the mechanism by which they synthesized DNA were known. Since then, much structural and kinetic data on DNA polymerases has emerged to suggest mechanistic explanations for the A-rule and the instructive and non-instructive behavior of lesions such as cis-syn dimers. Polymerases involved in the replication of undamaged DNA have highly constrained active sites that evolved to only accommodate the templating base and the complementary nucleotide and as a result are relatively intolerant of modifications that alter the size and shape of the nascent base pair. On the other hand, DNA damage bypass polymerases have much more open and less constrained active sites, which are much more tolerant of modifications. An otherwise instructional lesion would become non-instructional if it were unable to fit into the active site, and thereby behave transiently like an abasic site, leading to the insertion of whichever nucleotide is favored by the polymerase, generally an A. In this review, what is known about the active sites and mechanisms of replicative and DNA damage bypass polymerases will be discussed with regard to the A-rule and non-instructive behavior of lesions, typified by dipyrimidine photoproducts.

phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase

Abstract. A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechococcus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.

Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells.

In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.

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. We also found that the deamination of T(m)C and (m)CT CPDs is about 25-fold faster when flanked by Gs than by As, Cs or Ts in duplex DNA and appears to involve catalysis by the O6 group of guanine. In contrast, the first deamination of either C or (m)C in AC(m)CG with a flanking G was much slower (t(1/2) >250 h) and rate limiting, while the second deamination was much faster. The observation that C(m)CG dimers deaminate very slowly but at the same time correlate with C-->T mutation hotspots suggests that their repair must be slow enough to allow sufficient time for deamination. There are, however, a greater number of single C-->T mutations than CC-->TT mutations at C(m)CG sites even though the second deamination is very fast, which could reflect faster repair of doubly deaminated dimers.

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7.

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3′ thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5′ thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3′ base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.