Biomedical subjects

R D Camerini-Otero

Publications and source records attributed to R D Camerini-Otero.

At least 19 recordsLinked to original sources

PubMed2006

Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

Animals↗

PubMed2006

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages.

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.

Animals↗

PubMed2006

Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide.

Exposure of cells to ionizing radiation (IR) produces changes in the expression level of a large number of genes. However, less is known of gene-expression changes caused by local radiation exposure from radionuclides within cells. We studied changes in the genome-wide gene expression induced by decay of 125I incorporated into DNA as [125I]-iododeoxyuridine (125I-IUdR) in normal IMR-90 human lung fibroblasts and compared them with the changes produced by external gamma-radiation delivered at high (HDR) or low (LDR) dose rate. We found that more than 2000 genes were consistently up- or down-regulated following HDR and LDR gamma-radiation. The profiles of differentially expressed genes following HDR and LDR shared about 64% (up) and 74% (down) genes in common, with many genes identified as radiation-responsive for the first time. In contrast, in all only 206 genes changed their expression level in the 125I-IUdR-treated cells, even though the total number of DNA double-strand breaks (DSB) produced by 125I-IUdR exceeded that produced by the gamma-radiation. With few exceptions, the expression levels of 125I-IUdR-responsive genes were also altered following gamma-irradiation. Therefore, nuclear DNA-localized decays of 125I produce 10 times fewer differentially expressed genes than whole-cell exposure to gamma-radiation of comparable dose. These results suggest that the effect of IR on the changes in global gene expression depends on the distribution of energy depositions within the cell. In contrast to cell survival, DNA DSB may not be the major factor modulating changes in gene expression following irradiation.

Cell Line↗

PubMed2001

A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA.

DinI is a recently described negative regulator of the SOS response in Escherichia coli. Here we show that it physically interacts with RecA and prevents the binding of single-stranded DNA to RecA, which is required for the activation of the latter. DinI also displaces ssDNA from a stable RecA-DNA cofilament, thus eliminating the SOS signal. In addition, DinI inhibits RecA-mediated homologous DNA pairing, but has no effect on actively proceeding strand exchange. Biochemical data, together with the molecular structure, define the C-terminal alpha-helix in DinI as the active site of the protein. In an unusual example of molecular mimicry, a negatively charged surface on this alpha-helix, by imitating single-stranded DNA, interacts with the loop L2 homologous pairing region of RecA and interferes with the activation of RecA.

Amino Acid Sequence↗

PubMed2000

The homologous pairing domain of RecA also mediates the allosteric regulation of DNA binding and ATP hydrolysis: a remarkable concentration of functional residues.

Switching between the active (ATP and DNA bound) and inactive conformations of the homologous recombination RecA protein is regulated by ATP hydrolysis. First, we use the homologous pairing domain of RecA derived from its mobile loop L2 to show that the interaction of this random coil peptide with the gamma-phosphate of ATP results in a peptide beta-conformation similar to that previously shown to be induced by DNA binding. Next, we show that in the whole RecA protein two residues in this L2 domain, Gln194 and Arg196, are catalytic amino acid residues for ATP hydrolysis and functionally resemble the corresponding residues engaged in GTP hydrolysis by two distinct classes of G proteins. Finally, we show that the role of DNA and high salt in the stimulation of the ATPase of RecA is to stabilize this highly mobile region involved in hydrolysis. This is a role similar to that described for RGSs in the activation of the GTPase of heterotrimeric G proteins. Therefore, (i) a prototypical DNA-dependent ATPase and ATP-stimulated DNA-binding protein, RecA, and eukaryotic signaling proteins share common stereochemical regulatory mechanisms; and (ii) in a remarkable example of parsimony, loop L2 is a molecular switch that controls both ATP promoted DNA binding and pairing reactions and DNA stimulated ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed2000

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex.

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.

Base Sequence↗

PubMed2000

The mouse Spo11 gene is required for meiotic chromosome synapsis.

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.

Adenosine Triphosphatases↗

PubMed2000

Solution structure of DinI provides insight into its mode of RecA inactivation.

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.

Aspartic Acid↗

PubMed1999

Cloning, characterization, and localization of mouse and human SPO11.

Spo11 is a meiosis-specific protein in yeast that has been found covalently bound to DNA double-strand breaks (DSBs) during the early stages of meiosis. These DSBs initiate homologous recombination, which is required for proper segregation of chromosomes and the generation of genetic diversity during meiosis. Here we report the cloning, characterization, tissue expression, and chromosomal localization of both mouse and human homologues of Spo11. The putative mouse and human proteins are 82% identical and share approximately 25% identity with other family members. Northern blot analysis revealed testis-specific expression for both genes, but RT-PCR results showed ubiquitous expression of at least a portion of Spo11 in mouse. Human SPO11 was also detected in several somatic tissues. Mouse Spo11 was localized to chromosome 2H4, and human SPO11 was localized to chromosome 20q13.2-q13.3, a region amplified in some breast and ovarian tumors.

Alternative Splicing↗

PubMed1999

Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair.

The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational proficiency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identified as stringently required for recombinational events in bacterial cells. In addition, our findings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming filamentous beta-structures, we propose how the information from the mutant analysis might facilitate the use of a simplified amino acid alphabet to design beta-structure forming L2 peptides with improved RecA-like activities.

DNA Repair↗

PubMed1998

Dissociation kinetics of RecA protein-three-stranded DNA complexes reveals a low fidelity of RecA-assisted recognition of homology.

We determined that the incorporation of one mismatch into RecA mediated synaptic complexes between oligonucleotide single-stranded DNAs and target duplex DNAs destabilizes the complex by 0.8 to 1.9 kcal/mol. This finding supports our previous result, that RecA binding per se can significantly decrease the loss in free energy associated with mismatch incorporation even in the absence of ATP hydrolysis. We show that the specificity is mostly driven by the dissociation process. We found that the relative destabilization induced by different mismatches depends on their position. Thus, while there is a good correlation between the ranking order of mismatches at the 5' end of synaptic complexes and mismatches in heteroduplexes (D-loops), there is no correlation between the ranking order for mismatches at the 3' end and mismatches in various DNA structures. This difference between the 5' and 3' ends of synaptic complexes agrees well with the established 5' to 3' polarity of the strand exchange promoted by RecA protein. The lack of a correlation between mismatches at the 3' end of synaptic complexes and mismatches in D-loops suggests the intermediate is probably not a canonical protein-free D-loop.

DNA↗

PubMed1998

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Amino Acid Sequence↗

PubMed1998

Sequence-specific ligation of DNA using RecA protein.

A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.

Bacteriophage lambda↗

PubMed1997

An alteration in the structure of the minor groove of duplex DNA induced by the formation of an intermolecular d(GA)n:d(GA)n.d(TC)n triplex.

A simple homopolymeric purine:purine.pyrimidine, d(GA)n:d(GA)n.d(TC)n, triplex DNA was generated in the presence of magnesium ion at neutral pH. Hydrogen bonding of the N7 positions of guanines and adenines was investigated by 7-deaza base substitutions and protection from chemical modification by dimethyl sulfate (DMS). Chemical modification of this triplex with DMS revealed an unexpected triplex specific hypermodification in the N3 positions of the adenines in addition to the protection in the N7 positions of the guanines. The significance of this increase in the chemical accessibility of the N3 positions of adenines in the minor groove and possible relevance of dehydration to the mechanism of triplex formation and stability is discussed.

Adenine↗

PubMed1997

Photocross-linking of the NH2-terminal region of Taq MutS protein to the major groove of a heteroduplex DNA.

The MutS DNA mismatch repair protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25-49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Taq MutS protein.

Adenosine Triphosphatases↗

PubMed1997

RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide.

We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.

Base Composition↗

PubMed1997

The duplex DNA is very underwound in the three-stranded RecA protein-mediated synaptic complex.

BACKGROUND: The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear. RESULTS: We have exploited two approaches to study the structure of Escherichia coli RecA protein-mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I. The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn. We were able to show that the topology of the three-stranded complexes containing RecA is quite different from that expected for deproteinized D-loops. The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn. Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three-stranded helical structure. CONCLUSIONS: This work provides the first quantitative topological parameters for the RecA protein-mediated three-stranded synaptic complex. Furthermore, the structure of synaptic complexes is different from that of a simple D-loop.

Base Sequence↗

PubMed1996

Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA.

The molecular structure of the Escherichia coli RecA protein in the absence of DNA revealed two disordered or mobile loops that were proposed to be DNA binding sites. A short peptide spanning one of these loops was shown to carry out the key reaction mediated by the whole RecA protein: pairing (targeting) of a single-stranded DNA to its homologous site on a duplex DNA. In the course of the reaction the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a beta structure. These events probably recapitulate the underlying molecular pathway or mechanism used by homologous recombination proteins.

Amino Acid Sequence↗