The Walker Lab at MIT

Publications

Publications are available here in PDF format: Walker Lab publications

Research summary

Our research is concentrated in two major areas. The first concerns the regulation and mechanism of action of proteins involved in DNA repair and mutagenesis and in other cellular responses to DNA damage. The second deals with identification of the bacterial functions required for the development of nitrogen-fixing nodules on legumes and with the relationship between rhizobial functions required for nodule invasion/infection and mammalian pathogenesis.

Contents

DNA Repair and Mutagenesis in Bacteria:

One long-term focus of our lab has been understanding the SOS responses of E. coli to DNA damage, with a particular emphasis on the umuDC and dinB genes. UmuC (DNA pol V) and DinB (DNA pol IV) encode SOS-regulated Y Family DNA polymerases that can carry out translesion synthesis over DNA lesions; translesion synthesis is the mechanistic basis of most UV and chemical mutagenesis. The SOS genes are induced when the RecA-ssDNA nucleoprotein filaments that form after DNA damage facilitate an autodigestion of the LexA repressor. UmuD shares homology with the carboxyl-terminal domains of LexA and lambda repressor and undergoes a similar autodigestion facilitated by RecA-ssDNA nucleoprotein filaments to yield UmuD'. In a temporally ordered process, the UmuD2C proteins act in a bacterial DNA damage checkpoint that promotes cell survival while the UmuD'2C products then carry out translesion synthesis. We recently discovered that E. coli DinB and its mammalian and archaeal orthologs are 10-15X better polymerases when replicating lesions similar to N2-furfuryl dG than when replicating dG. Remarkably, a single amino acid change can separate DinB's translesion synthesis capability from its ability to replicate normal DNA.

Our recent work has revealed that UmuD and RecA not only regulate UmuC, but also play unanticipated roles in regulating DinB. By capping off DinB's open active site, they suppress the -1 frameshift mutagenesis caused by template bulging. Our further discovery that UmuD2 and UmuD'2 are intrinsically disordered proteins has revealed an underlying molecular principle that enables these small proteins to interact with multiple partners, including three subunits of the replicative DNA polymerase, as they exert their regulatory roles.

Our efforts to understand cellular responses to DNA damage within the complex architecture of a living cell have been carried out in collaboration with Alan Grossman. We discovered that DNA replication is required for formation of RecA-GFP foci after exposure to DNA-damaging agents. Our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. MutS homologs function in several cellular pathways including mismatch repair, the process by which mismatches introduced during DNA replication are corrected. We have recently shown that the C-terminus of Bacillus subtilis MutS is necessary for an interaction with beta-clamp processivity clamp and that this interaction is required for MutS-GFP focus formation in response to mismatches.

On-going research includes studies of DinB mechanism, function, and control; novel global aspects of SOS control; and the complex physiological responses of cells to depletion of deoxyribonucleotides.

DNA Repair and Mutagenesis in Eukaryotes:

Humans have four Y Family translesion DNA polymerases including the Xeroderma pigmentosum variant gene product (pol eta), which can copy accurately over cyclobutane thymine-thymine dimers. S. cerevisiae has two, pol eta and Rev1, while S. pombe additionally encodes a dinB ortholog (pol kappa). Various observations suggest that, like UmuC and DinB, the action of these DNA polymerases is controlled by a complex set of protein-protein interactions and we have initiated an effort to understand the molecular basis of the control of these proteins in yeast and humans. We recently discovered that S. cerevisiae Rev1 (required for UV and chemical mutagenesis in all eukaryotes) is dramatically cell-cycle regulated (50 fold higher in G2/M than in G1 and S). This observation suggests that mutagenic TLS occurs at gaps left behind after DNA replication rather than at the replication fork itself.

Although Rev1's capacity as a DNA polymerase is limited to inserting dC's, it plays a critical role by recruiting other translesion DNA polymerases including pol zeta (Rev3/Rev7). We have identified novel and hitherto unrecognized conserved motifs that play an essential role in REV1-dependent mutagenesis in S. cerevisiae and have shown that a minimal C-terminal fragment of Rev1 containing these highly conserved motifs is sufficient to interact with Rev7.

On-going projects include investigating the molecular basis of Rev1 cell-cycle control; extending our studies of Rev1 function to mammals in collaboration with Michael Hemann; exploring the UBZ ubiquitin-binding domain of S. cerevisiae pol eta; and analyzing the function and control of the S. pombe dinB ortholog.

Rhizobium functions required for nodule invasion and chronic infection; their relationship to functions utilized by mammalian pathogens:

Sinorhizobium meliloti establishes a symbiosis with alfalfa in which it converts atmospheric nitrogen to ammonia. We are studying the mechanisms by which S. meliloti invades the nodules that it elicits on roots and the functions it requires to establish the chronic intracellular infections of host cells necessary for the symbiosis.

Our extensive genetic analyses of the Sinorhizobium-legume symbiosis led to the discovery that these rhizobia synthesize structurally different, but functionally equivalent, acidic exopolysaccharides that play an essential role in nodule invasion on alfalfa by enabling infection thread initiation and extension. We determined the complete pathway for succinoglycan biosynthesis and the multiple mechanisms for generating the low molecular weight derivatives of succinoglycan and other exopolysaccharides that appear to be the symbiotically active forms. Our recent microarray analysis indicates that appropriate symbiotically active exopolysaccharides act as signals to plant hosts to initiate infection thread formation and that, in the absence of this signal, plants terminate the infection process, perhaps via a defense response.

We discovered that the plant symbiont S. meliloti and the mammalian pathogen Brucella abortus share common molecular functions that are required for the chronic intracellular infections of eukaryotic cells that underlie their respective biological roles. For example, we found that the bacA gene, whose function is required for S. meliloti chronic intracellular infection of plant cells inside the nodule, is present in B. abortus as well, where is required for chronic pathogenesis in BALB/c mice. The limited similarity of BacA to the human X-linked adrenoleukodystrophy protein (a peroxisomal transporter of very long chain fatty acids) led us to the discovery that BacA affects the modification of lipid A by very long chain fatty acids (C28/C30) in both S. meliloti and B. abortus.

We have recently shown that the S. meliloti BluB protein, whose absence results in a symbiotic deficiency, carries out what had been the missing step in vitamin B12 biosynthesis, the synthesis of the "lower ligand" dimethylbenzimidazole (DMB). BluB is a new class of enzyme, a flavin destructase that cannibalizes reduced FMN to make DMB. Crystallographic analysis revealed the relationship between oxidoreductases, which use FMN as a cofactor, and BluB, which uses FMN as a substrate.

Ongoing projects include investigations of the details of BluB function and the molecular nature of the B12 requirement for symbiosis; an examination of a master regulator of symbiosis (CbrA) that is also a principle regulator of the bacterial cell cycle in S. meliloti; characterization of a highly conserved gene found in all bacteria whose product is critical for symbiosis; and the role of non-homologous end-joining (NHEJ) in symbiosis.