Lab Photo 2017

Research Overview

What are replication-transcription conflicts?

Bacteria (and most other organisms) have a common requirement for accurate, timely, and faithful DNA replication. Yet a wide variety natural impediments are known to physically slow or stall DNA replication forks, reducing fitness and increasing mutation rates. Examples include chemical lesions, broken strands, and tightly bound proteins. Much of our interest lies in identifying such obstacles, and determining what happens when DNA replication forks encounter them. In particular, we have found that transcription is the most significant impediment to DNA replication. Our single molecule studies of replisome dynamics show that active transcription collapses the replication fork and the replisome multiple times every cell cycle, increasing mutation rates and strand breakage, and cutting the average rate of DNA replication in half. Hence replication-transcription conflicts are a major and fundamental problem for bacteria.

Our lab uses a variety of cutting edge tools to study replication-transcription conflicts. These include in vivo single molecule microscopy, deep sequencing, bioinformatics, and lab-based experimental evolution. We pair these new technologies with classical genetics and molecular biology techniques, typically using the model bacterium Bacillus subtilis. This naturally competent bacteria grows quickly, is genetically tractable, and a deletion collection is available. However, in recent years we have also branched out into pathogens such including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Salmonella enterica.

How are replication-transcription conflicts resolved?

Replication and transcription are not temporally separated. This leads to collisions between the two machines as they move along the DNA. Because genes can be encoded on either the Watson or the Crick strand, collisions can occur either head-on or co-directionally. Though head-on collisions are far more severe than co-directional collisions, both can promote strand breakage and replisome collapse, requiring the action of replisome restart proteins. To maximize fitness and protect the genome from unwanted instability caused by these inevitable encounters, cells have developed mechanisms to resolve conflicts. Thus far, we have identified several critical conflict resolution mechanisms. These include the accessory helicase PcrA, which appears to bolster the strength of the replisome and allow it to advance through even the most heavily transcribed head-on genes, and RNase HIII, an enzyme that degrades the RNA portion of stable DNA:RNA hybrids. We found that these R-loops form specifically at head-on (but not at co-directional) conflict regions, permanently blocking the replication fork and killing the cell if left unresolved. Interestingly, our discovery of the orientation-specific nature of R-loop formation and the need for RNase HIII coincided with a similar discovery in human cells. The consistency between our results in bacteria and findings in higher organisms demonstrate that replication-transcription conflicts and their downstream consequences are similar throughout all life.

How do replication-transcription conflicts influence genome organization?

Replication and transcription are not temporally separated. This leads to collisions between the two machines as they move along the DNA. Because genes can be encoded on either the Watson or the Crick strand, collisions can occur either head-on or co-directionally. Though head-on collisions are far more severe than co-directional collisions, both can promote strand breakage and replisome collapse, requiring the action of replisome restart proteins. To maximize fitness and protect the genome from unwanted instability caused by these inevitable encounters, cells have developed mechanisms to resolve conflicts. Thus far, we have identified several critical conflict resolution mechanisms. These include the accessory helicase PcrA, which appears to bolster the strength of the replisome and allow it to advance through even the most heavily transcribed head-on genes, and RNase HIII, an enzyme that degrades the RNA portion of stable DNA:RNA hybrids. We found that these R-loops form specifically at head-on (but not at co-directional) conflict regions, permanently blocking the replication fork and killing the cell if left unresolved. Interestingly, our discovery of the orientation-specific nature of R-loop formation and the need for RNase HIII coincided with a similar discovery in human cells. The consistency between our results in bacteria and findings in higher organisms demonstrate that replication-transcription conflicts and their downstream consequences are similar throughout all life.

Blocking evolution to prevent antibiotic resistance development

We recently discovered that head-on conflicts drive up mutation rates. This finding could potentially explain a second observation - that in the wild, head-on genes evolve at a faster rate (on average) than their co-directional counterparts. Our lab has made major progress towards identifying and characterizing some of the underlying mechanisms that actively increase mutation rates after replication-transcription collisions. In particular, we have identified a new class of proteins that we are terming "evolvability factors". Our new data show that these proteins promote rapid adaptation to new environmental stresses including exposure to antibiotics. Using new state of the art high-throughput lab-based evolution experiments and bioinformatics analyses, we are currently investigating the molecular mechanisms underlying these adaptive changes.