Biomedical subjects

Marie-Pierre Golinelli

Publications and source records attributed to Marie-Pierre Golinelli.

4 recordsLinked to original sources

PubMed2003

Secondary structure in the nucleic acid affects the rate of HIV-1 nucleocapsid-mediated strand annealing.

We studied the effects of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein on the kinetics of annealing of nucleic acids using model substrates derived from the 3' end of the HIV-1 minus-strand strong-stop DNA (-sssDNA). We used HIV-1 reverse transcriptase (RT) to monitor the annealing reaction. Using several different DNA primers and acceptor oligonucleotides, we found that the rate of annealing increased with the size of the complementary region of the primer and the acceptor strands and decreased when secondary structures could be formed in either the primer or the acceptor strands. The secondary structure had a larger effect on the rate of annealing if the secondary structure extends to the 3' end of the nucleic acid(s). NC protein reduced the rate of annealing between strands with short homologies. NC had no major effect on the rate of annealing when there were at least 13 bases of complementarity between the primer and the acceptor strands and neither strand could form a stable secondary structure. NC increased the rate of annealing when the primer and/or the acceptor strand could form a secondary structure in the region of complementarity. When two strands were in competition as acceptors in an annealing reaction, the specificity of the annealing was determined by the length of the complementarity between the primer and the acceptor strands, the presence or the absence of secondary structures in the primer and/or the acceptor strand, and the presence or the absence of NC in the reaction. This suggests that NC facilitates strand transfer where the nucleic acids have considerable secondary structure (for example, the first strand transfers for viruses whose genomes have considerable secondary structure at their 3' ends). However, NC also appears to increase the fidelity of recombination by reducing strand transfers between segments that have limited complementarity.

Base Sequence↗

PubMed2002

Nontemplated nucleotide addition by HIV-1 reverse transcriptase.

We studied the kinetics of nontemplated nucleotide addition by the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) using model substrates derived from the 3' end of HIV-1 minus-strand strong-stop DNA. The addition of a nontemplated nucleotide was highly dependent on the nature of the base (fastest addition with dATP), type of nucleoside, and pH of the reaction buffer. The salt concentration, presence or absence of nucleocapsid protein, and nature of the blunt-ended duplex (DNA/DNA versus RNA/DNA) had only limited effects. The efficiency and base specificity were strongly affected by the sequence at the 3' end of the blunt-ended duplex. In every case, nontemplated nucleotide addition was much slower than templated polymerization. The K(d) for the incoming dNTP with an RT bound to a blunt-ended duplex was at least 1000-fold higher than with a duplex with a template overhang. At concentrations normally found in vivo, ATP can compete with dNTPs for binding to the polymerase active site and reduce the efficiency of nontemplated nucleotide addition. Although a stable ternary complex RT/DNA/dNTP could be readily detected by gel retardation assays if the DNA had a template overhang, stable ternary complexes were not observed with a blunt-ended duplex substrate. At in vivo concentrations of dNTPs (5-10 microM), nontemplated nucleotide addition occurred, but it was very inefficient and the rate of nontemplated polymerization is at least 10000-fold slower than the rate of templated polymerization. We could conclude that, in vivo, the unfavorable binding of the incoming dNTP, low concentration of dNTPs, the presence of a large concentration of ATP, and the inability to form a stable ternary complex prior to the polymerization step collaborate to reduce the efficiency of nontemplated nucleotide addition.

Adenosine Triphosphate↗

PubMed2002

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster.

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.

Amino Acid Sequence↗

PubMed2002

Nontemplated base addition by HIV-1 RT can induce nonspecific strand transfer in vitro.

After minus-strand strong-stop DNA (-sssDNA) synthesis, the RNA template is degraded by the RNase H activity of reverse transcriptase (RT), generating a single-stranded DNA. The genomes of some retroviruses contain sequences that could lead to self-priming of their minus signsssDNA. Self-priming was prevented by annealing a DNA oligonucleotide to the 3' end of model DNAs that corresponded to the 3' ends of the -sssDNAs (-R ssDNA) from human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and human T-cell leukemia virus type 1 (HTLV-1) but nonspecific strand transfer to ssDNA molecules in solution was induced in vitro (Golinelli and Hughes, 2001). This nonspecific strand transfer involved the addition of a nontemplated base to the 3' end of -R ssDNAs that was part of a blunt-ended duplex. In the case of HIV-2 -R ssDNA, A and C were added more efficiently than G and T. Strand transfer to ssDNA in solution occurred only if the nontemplated base could form a basepair with the last base at the 3' end of the ssDNA. If there was a mismatch, strand transfer did not occur. There was no detectable strand transfer to internal sites in the target ssDNA except to the second position from the 3' end of the DNA acceptor when the sequences at the 3' ends of the two DNAs allowed the formation of two basepairs. The nontemplated base addition and the one-basepair strand transfer were both affected by the salt concentration in the reaction, the nature of the reverse transcriptase (HIV-1 versus Moloney murine leukemia virus), and the sequence at the 3' end of -R ssDNA. NC reduced the efficiency of nonspecific strand transfer in vitro, suggesting that NC may have a role in reducing nonspecific strand transfer in vivo.

Base Pairing↗