Biomedical subjects

Paul D Chastain

Publications and source records attributed to Paul D Chastain.

9 recordsLinked to original sources

PubMed2006

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1.

An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA. The promoter region of FMR2 in the FRAXE site (approximately 600 kb away, in chromosome band Xq28) also includes an origin of replication, as previously described [Chastain II, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., Kaufman, D.G., 2006. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869-872]. FMR1 transcripts were detected in foreskin and male fetal lung fibroblasts, while FMR2 transcripts were not. However, both FMR1 and FMR2 were found to replicate late in S phase (approximately 6 h into the S phase of normal human fibroblasts). The position of the origin of replication relative to the CGG repeat, and perhaps the late replication of these genes, might be important factors in the susceptibility to triplet repeat amplification at the FRAXA and FRAXE sites.

Cell Line↗

PubMed2006

Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.

Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication.

Bacteriophage T4↗

PubMed2006

Nonrandom AP site distribution in highly proliferative cells.

Reactive oxygen species (ROS) and the oxidative DNA damage they produce [e.g., 8-oxo-guanine and apurinic/apyrimidinic (AP) sites] have been linked to the pathogenesis of several age-related and chronic diseases. The basal number of AP sites measured in DNA by immuno-slot-blot analysis ranges from 70,000 to 100,000 per genome. We used electron microscopy to determine how AP sites were distributed in isolated DNA fibers from fresh calf thymus and HeLa cell cultures. We observed that AP sites were not equally distributed throughout all the fibers. A small percentage of the analyzed DNA fibers contained a disproportionate amount of the total AP sites in nonrandom groups of 10 to >30 closely spaced in a small region (e.g., 20 AP sites in a 6 kb length of DNA). This finding suggests that genomic sites may differ in their vulnerability to ROS damage, perhaps because of local chromatin structure. Nonrandom AP site formation also suggests that the detrimental effects of ROS in the development of disease may be related not simply to the total number of AP sites present but to how AP sites are distributed along a DNA fiber and, perhaps, to the genomic sites affected.

Amino Acid Sequence↗

PubMed2006

Checkpoint regulation of replication dynamics in UV-irradiated human cells.

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene.

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene. Identification of this origin may aid in the investigation of the mechanism of trinucleotide repeat expansion and its effect on FMR2 expression. In addition, knowledge of the chromosomal locations and sequence characteristics of both early and late origins of DNA replication, such as the one described in this report, will facilitate studies of the molecular determinants of the time of activation of different origins of replication and allow us to refine our insights concerning origin inactivation in response to the DNA damage-induced intra-S checkpoint.

Cell Line↗

PubMed2004

Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer.

The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.

Adenosine Triphosphate↗

PubMed2003

Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins.

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.

Bacteriophage T4↗

PubMed2002

Sticky DNA, a long GAA.GAA.TTC triplex that is formed intramolecularly, in the sequence of intron 1 of the frataxin gene.

Friedreich's ataxia is caused by the massive expansion of GAA.TTC repeats in intron 1 of the frataxin (X25) gene. Our prior investigations showed that long GAA.TTC repeats formed very stable triplex structures which caused two repeat tracts to adhere to each other (sticky DNA). This process was dependent on negative supercoiling and the presence of divalent metal ions. Herein, we have investigated the formation of sticky DNA from plasmid monomers and dimers; sticky DNA is formed only when two tracts of sufficiently long (GAA.TTC)(n) (n = 59-270) are present in a single plasmid DNA and are in the direct repeat orientation. If the inserts are in the indirect (inverted) repeat orientation, no sticky DNA was observed. Furthermore, kinetic studies support the intramolecular nature of sticky DNA formation. Electron microscopy investigations also provide strong data for sticky DNA as a single long triplex. Hence, these results give new insights into our understanding of the capacity of sticky DNA to inhibit transcription and thereby reduce the level of frataxin protein as related to the etiology of Friedreich's ataxia.

Cloning, Molecular↗

PubMed2002

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins.

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.

Bacteriophage T7↗