5-fluorouracil (5-FU) is widely used in the treatment of cancer. Over the past 20 years, increased understanding of the mechanism of action of 5-FU has led to the development of strategies that increase its anticancer activity. Despite these advances, drug resistance remains a significant limitation to the clinical use of 5-FU. Emerging technologies, such as DNA microarray profiling, have the potential to identify novel genes that are involved in mediating resistance to 5-FU. Such target genes might prove to be therapeutically valuable as new targets for chemotherapy, or as predictive biomarkers of response to 5-FU-based chemotherapy.

5-Fluorouracil: mechanisms of action and clinical strategies

Replication protein A [RPA; also known as replication factor A (RFA) and human single-stranded DNA-binding protein] is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination. RPA homologues have been identified in all eukaryotic organisms examined and are all abundant heterotrimeric proteins composed of subunits of approximately 70, 30, and 14 kDa. Members of this family bind nonspecifically to single-stranded DNA and interact with and/or modify the activities of multiple proteins. In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage). Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell. RPA may also have a role in modulating gene expression.

REPLICATION PROTEIN A: A Heterotrimeric, Single-Stranded DNA-Binding Protein Required for Eukaryotic DNA Metabolism

The structure

http://www.jbc.org/content/279/17/16895.full.pdf

DNA Replication Fidelity

The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring-shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring-shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA–protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring-shaped ATPases.

AAA+ superfamily ATPases: common structure--diverse function.

Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate.

Coupling of a Replicative Polymerase and Helicase: A τ–DnaB Interaction Mediates Rapid Replication Fork Movement

The tau and gamma subunits of DNA polymerase III holoenzyme are both products of the dnaX gene. Since tau and gamma are required as stoichiometric components of the replicative complex, a mechanism must exist for the cell to coordinate their synthesis and ensure that both subunits are present in an adequate quantity and ratio for assembly. We have proposed that gamma is produced by a translational frameshift event. In this report, we describe the use of dnaX-lacZ fusions in all three reading frames to demonstrate that gamma, the shorter product of dnaX, is generated by ribosomal frameshifting to the -1 reading frame of the mRNA within an oligo(A) sequence that is followed by a sequence predicted to form a stable secondary structure. Immediately after frameshifting a stop codon is encountered, leading to translational termination. Mutagenesis of the oligo(A) sequence abolishes frameshifting, and partial disruption of the predicted distal secondary structure severely impairs the efficiency. Comparison of the expression of lacZ fused to dnaX distal to the site of frameshifting in the -1 and 0 reading frames indicates that the efficiency of frameshifting is approximately 40%.

https://www.pnas.org/content/pnas/87/10/3713.full.pdf

The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting.

DNA Polymerase III Holoenzyme .... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Polymerase III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Polymerase III' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Polymerase 111* . . Isolated Holoenzyme Subunits . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Relationship of DNA Polymerase II and Complex Polymerase Forms .... .. PROCESSIVITY OF DNA POLYMERASE III FORMS . . . . . . . . ..... . . . . . . ... . . . . . . ........ . STAGES IN lHE DNA POLYMERASE III HOLOENZYME-CATALYZED REACTION . ........ . .. . . ... .

Charles S. McHenry

Papers

Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate.

Coupling of a Replicative Polymerase and Helicase: A τ–DnaB Interaction Mediates Rapid Replication Fork Movement

The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting.

DNA polymerase III holoenzyme of Escherichia coli.

Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription.