The dynamics of accessory proteins in DNA replication and DNA repair.

摘要

DNA replication is responsible for the maintenance of life, ensuring the integrity of the genome from one generation to the next. However, all cells are exposed to a variety of natural and synthetic DNA-damaging agents, which can interfere with DNA replication. Cells have developed, therefore, their own DNA repair systems. It is extremely important to understand the mechanisms of DNA replication and repair, in order to determine how cells survive DNA damage. However, there is still little known about the functional dynamics and conformation of some of the accessory proteins in these systems.;In Escherichia coli, UmuD and UmuDO' are DNA-damage responsive, DNA polymerase management proteins, both generated from the umuD gene. UmuDO' is the cleaved form which is missing 24 residues from the N-termini of UmuD. Full-length UmuD inhibits mutagenesis, whereas UmuDO' facilitates mutagenesis. Hydrogen exchange mass spectrometry (HX MS) was performed to measure the conformational differences among UmuD, UmuDO', and UmuD3A (an uncleavable mutant). Our results reveal that these three forms of the umuD gene products are flexible, especially the N-terminal arms of UmuD that play a significant role in the conformational transition upon cleavage. The residues that are connected to the N-terminal arms display more deuterium incorporation in UmuDO' and UmuD3A than in UmuD. The observations suggest that the noncleavable UmuD3A mimics the cleaved form (UmuDO') because in both cases the arms are relatively unbound from the globular domain.;By far, the best characterized interactions of UmuD or UmuDO' with the replisome are the interactions with the β clamp of E. coli DNA pol III. The β clamp is a ring-shaped homodimer that makes highly processive DNA replication possible. The dynamics and conformational changes among the β clamp, a monomeric variant, and the β clamp/UmuD complex were determined by HX MS and compared. Our results reveal that the β clamp is not a static closed ring in solution. The three domains of the β clamp showed different dynamics though they share nearly identical tertiary structure. Several peptides in Domain I were observed to be much more dynamic than the other domains and displayed partial local unfolding, so called EX1 kinetics, with a half-life of about 4 h. In the monomeric variant of the β clamp, partial unfolding was much faster and additional regions of Domain III also underwent partial unfolding with a half-life of about 1 h. Our data suggest that the dimer interface of the β clamp may open spontaneously by Domain I dissociating from Domain III of its partner protomer.;The exciting findings on the dynamics of β clamp raised our interest in studying other clamps. The functions and tertiary structure of processivity clamps are highly-conserved in all organisms. Four well-characterized clamps from different species (T4 bacteriophage gp45, yeast PCNA, two euryarchaeon PCNAs) were selected to compare their dynamics by HX MS. Our data indicate that the processivity clamps showed remarkably different dynamics in solution. The dynamics order based on the local unfolding was found to be: T4 gp45 > bacteria β clamp > yeast PCNA, TK 0582 > TK 0535, with T4 gp45 being the most dynamic. We conclude that high tertiary structure conservation of proteins and protein domains does not necessarily translate to high conservation of dynamics and therefore methods like HX MS must be utilized to measure protein dynamics.