We have two ultimate goals. Our first goal is to understand the biochemistry of L1 replication using one of the two L1-encoded proteins, ORF1p, as an entry point and determining its role and mechanism of action. The second protein, ORF2p, acts as the L1 replicase, a function that is fairly well established. However the role of ORF1p, which is an unusual trimeric nucleic acid chaperone, remains enigmatic. Therefore, we feel that the key to the mechanism of L1 replication is how this protein functions as a nucleic acid chaperone, and how either the properties that endow the protein with chaperone activity or the chaperon activity itself support L1 replication. We use a combination of evolutionary analysis (to frame our biochemical questions) and biochemical, structural, and cellular biological approaches to study ORF1p function.
Our second goal is to understand the determinants of the neutral mutation rate (i.e., the rate at which base substitutions accumulate). Base substitutions are the most common class of mutations and an important source of genetic novelty and genetic diseases. Thus, determining the factors that affect the neutral mutation rate is of paramount significance to both biology and medicine. Our bioinformatic analysis of nonfunctioning L1 DNA fossils showed that the rate at which base substitutions accumulate in these sequences is directly proportional to their CpG content. More specifically, we found that fossil L1 sequences with a higher CpG content accumulate mutations at non-CpG sites faster than those with lower CpG content. As CpGs are mutational hot spots in mammals and thus foci for DNA repair, we suggested that DNA repair itself may be mutagenic to flanking normal DNA. We have experimentally demonstrated a mutagenic effect of DNA repair in vivo and are now determining both the parameters and biochemical basis for it.
L1 retrotransposons are autonomous genetic elements that have been replicating and evolving in mammalian genomes for more than 80 million years. L1 replicates by copying its RNA transcript—and that of other genes—into genomic DNA. By now, L1-generated insertions account for approximately 50 percent of mammalian DNA. These insertions represent an unparalleled source of DNA fossils that provide a genetic record of its host. L1 activity persists in modern mammals, generating new inserts that can inactivate or alter gene activity and cause genetic rearrangements, which reduce the genetic fitness of humans. Despite its dominant past and ongoing effects on the structure and function of mammalian genomes, little is known about the regulation of L1 activity, the biochemistry of L1 replication, or how L1 interacts with its host, which represents a major gap in our understanding of mammalian biology.
We pursue two major goals. The first major goal is understanding the biochemistry of L1 replication. L1 encodes two proteins, ORF1p and ORF2p, which are essential for its replication. ORF2p is the replicase, but the role of ORF1p, which is an unusual trimeric nucleic acid chaperone, is particularly enigmatic. We think that understanding the mechanism and role of ORF1p chaperone activity in L1 replication is essential to understanding the L1 replication cycle. We use evolutionary, structural, and biochemical analyses to study ORF1p. This knowledge is also applied to nucleic acid chaperones in general, a class of enzymes involved in diverse biochemical pathways, including viral replication and those that cause human disease.
Our second major goal is determining the factors that affect the mutation rate. Our studies on L1 DNA fossils suggested that DNA repair may be mutagenic to normal flanking DNA. We use bioinformatic and molecular biological approaches to study this concept and have recently verified it experimentally. That DNA repair can be mutagenic has profound implications for understanding the determinants of the neutral mutation rate; for using this rate in evolutionary studies; and for using analyses that rely on sequence conservation, or lack thereof, as a proxy for selection. This project is now a major focus in our laboratory.