Eucaryotic DNA replication complex: study of structure and function using the affinity modification technique.

Eucaryotic DNA replication complex is now one of the most intensively studied subjects of molecular biology and biochemistry. In addition to detailed studies on the structures and functions of individual DNA polymerases involved in this process, other enzymes and protein factors are also given much attention. The structures and functions of proteins in the replication complexes are studied by various approaches, including X-ray diffraction analysis.

Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains.

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein-DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5'-protruding ssDNA tails of 10, 20 and 30 nt.

[The eukaryotic replication complex and its affinity modification analysis].

Replication of eukaryotic DNA is driven by a protein complex, in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases alpha, delta, and epsilon involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is in an interplay with DNA replication.

[Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap].

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA.

[Interaction of human replication protein A with DNA-duplexes, containing gaps of varying sizes].

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3' end of a gap-flanking oligonucleotide with DNA polymerase beta, and an oligonucleotide containing a 5'-photoreactive group was chemically synthesized.

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs.

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5'-flap strands of the branched DNA structure and possesses 5'-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides.

[Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins].

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain.

Polarity of human replication protein A binding to DNA.

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER.

Human breast milk immunoglobulins G hydrolyze nucleotides.

Catalytically active antibodies, abzymes, appear in the blood of mammals immunized with the analogs of transition state or in the case of autoimmune diseases. Until recently, it was not shown whether abzymes exist in the blood of apparently healthy subjects. We have discovered that secretory IgA (sIgA) from the milk of healthy mothers catalyze phosphorylation of a variety of proteins and that IgG can hydrolyze DNA and RNA.