Our group is focused in the study of the protein-primed DNA replication mechanism, using as model the Bacillus subtilis phage °29. The development of an in vitro replication system with purified proteins and DNA from the phage laid the foundations of the protein-primed mechanism of DNA replication and made °29 a paradigm for studying this process. Although the protein-priming mechanism of DNA replication has been studied extensively in vitro, the underlying mechanisms governing the in vivo organization of the proteins involved in this process was poorly understood. Recently, we have provided insights into the in vivo organization of proteins involved in °29 DNA replication by showing that the bacterial actin-like MreB cytoskeleton, playing important roles in several cellular processes, is required for efficient DNA replication of the distantly related phages °29, SPP1 and PRD1. Components of the °29 DNA replication machinery, such as the DNA polymerase, are redistributed in peripheral helix-like structures in a cytoskeleton-dependent way. In addition, we have determined that the °29 terminal protein directs early organization of °29 DNA replication at the bacterial nucleoid.
From the biotechnological point of view we have improved the amplification performance of °29 DNA polymerase by fusion of DNA binding motifs. In addition, by changing the amplification conditions we have reduced 103 to 104-fold the amount of DNA that can be amplified. We have also developed a DNA amplification system primed by the TP based on the minimal replication origins of °29 DNA.
Bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Recently, we have identified an inhibitor (protein p56) of the B.subtilis Uracil DNA Glycosylase (UDG) encoded by bacteriophage °29. In vitro experiments showed that protein p56 blocks the DNA-binding ability of UDG, suggesting that p56 could be a novel naturally occurring DNA mimicry. We have suggested that inhibition of the host UDG by protein p56 is likely a defense mechanism developed by °29 and °29-related phages, which also encode p56-like proteins, to prevent the damaging action of the BER pathway if uracils arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA. We have also shown that protein p56 inhibits the DNA-binding capacity of UDG. In collaboration with Dr. Juan Luis Asensio (Instituto de QuÝmica Orgßnica, CSIC) the NMR structure of p56 has been determined. Amino acids, both in p56 and in UDG, involved in UDG inhibition were determined by site-directed mutagenesis.
On the other hand, we have shown that Bacillus subtilis gene yshC encodes a family X DNA polymerase whose biochemical features suggest a role during DNA repair processes. The determination of the phenotype of cells devoid of such a gene under conditions of DNA damage will provide relevant information on the role of this group of DNA polymerases widely distributed from viruses to higher eukaryotes.
For further information:
: modeling of the chimerical DNA polymerase. The figure represents the structural model of a (HhH)2 domain (colored in cyan) joint through a linker peptide (in dark blue) to the C-terminus of °29 DNA polymerase (colored in violet).
: °29 terminal protein (TP) localizes at the bacterial nucleoid. YFP, DAPI staining and merged images of typical Bacillus subtilis cells expressing a xylose-induced YFP-TP fusion protein analyzed 30 min after addition of the inductor.