Physical Biochemistry Of Macromolecules

与Samuel Wilson博士（NIEHS）的实验室就DNA转录起始和DNA修复中涉及的蛋白质进行了合作研究。这些工作包括DNA连接酶I与复制蛋白、增殖细胞核抗原（PCNA）之间的相互作用；XRCC1（1-183）蛋白与DNA聚合酶- β及其子结构域的相互作用机制；AP内切酶与DNA、DNA聚合酶与DNA以及两者与DNA的相互作用。目前已经完成了DNA酶的两个方面的研究，即PCNA的寡聚热力学和PCNA与组氨酸标记和未标记的FEN-1的相互作用。两份手稿正在准备中。预计这项工作将在明年继续进行。

Collaborative studies with the laboratory of Dr. Samuel Wilson (NIEHS) on proteins involved in DNA transcription initiation and in DNA repair have continued. These have included work on the interactions between DNA Ligase I and the replication protein, proliferating cell nuclear antigen (PCNA); the interaction mechanisms between the XRCC1(1-183) protein and DNA polymerase-beta and its subdomains; the interactions between AP endonuclease and DNA, DNA polymerase-beta and DNA, and both together with DNA. Research has been completed on two aspects of the studies on DNA enzymes, the thermodynamics of oligomerization of PCNA and the interaction of PCNA with histidine-tagged and untagged FEN-1. Two manuscripts are in preparation. It is anticipated that this work will continue in the coming year. In a collaborative study with the laboratory of Dr. Carl Wu (NCI), we have demonstrated that the proteins Chz1, Htz1, and H2B form a reversible complex with a 1:1:1 stoichiometry. This complex appears to be a novel histone chaperone that has specificity for H2AZ and can deliver the histone variant for SWR1-dependent histone replacement in eukaryotic cells. A manuscript entitled "Chz1, a Novel Nuclear Chaperone for Histone H2AZ" has been published in the journal "Molecular Cell." In a collaborative study with the laboratory of Dr. Daniel Appella (NIDDK), we have been studying the self-associative properties of a synthetic oligonucleotide (referred to here as DNA) and a similar molecule with the same bases attached to a polypepetide backbone (referred to here as PNA). We have demonstrated that the DNA is extremely thermally stable and undergoes a reversible dimer-tetramer self-association with a mean free energy change of -6.88 kcal/mol that has very little temperature dependence and is virtually totally of enthalpic origin. This is entirely consistent with its structure. We find that the PNA has significantly less stability than the DNA, showing signs of degradation at 34 deg. C. It undergoes a monomer-tetramer self-association with a mean free energy change of -7.7 kcal/mol, a weaker self-association than that exhibited by the DNA, since that is for a dimer-tetramer association and the PNA undergoes a monomer-tetramer association. The PNA self-association exhibits little temperature dependence and also is virtually totally of enthalpic origin, again consistent with its structure. A mixture of the DNA and the PNA associates very strongly with a mean free energy change of -19.6 kcal/mol. However, it exhibits unusual temperature dependency and preliminary analysis gave unexpected values for the thermodynamic parameters. The mean value of the change in enthalpy was -3.4 kcal/mol; the mean value for the change in entropy was 59 cal/mol/K; and the mean value for the specific heat content was -2.5 kcal/mol/K. Thus, the values of the change in free energy appear to be essentially a case of enthalpy-entropy compensation. While this is quite common in protein-protein association, it is quite unexpected here and will require considerable additional studies to explain. This is in marked contrast to the thermodynamic behavior of either the DNA or the PNA. Additionally, at temperatures of 28 deg. C and above, no heterotetramer was detectable. Whether this is due to the initiation of degradation of the PNA or the formation of some structure other than heterotetramer remains to be determined. This problem is of considerable biological interest since the PNAs appear to be a particularly specific means for the inhibition of DNA transcription and may have a significant role in the control of malignancy. The results of a collaboration with the laboratory of Dr. Ettore Appella (NCI), performed several years ago, entitled "Unfolding, Aggregation and Amyloid Formation by the Tetramerization Domain From Mutant p53 Associated With Lung Cancer" has been published in the journal "Biochemistry."