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Research Overview

Understanding DNA Structure and Function

The Mirkin laboratory uses a variety of models to understand DNA function and integrity in normal physiology and in several disease states.

Replication and Expansion of Simple DNA Repeats

Uncontrollable expansions of trinucleotide repeats lead to more than two dozens human hereditary neurological disorders, including Fragile X mental retardation, Huntington's disease, myotonic dystrophy, Friedreich's ataxia, etc. The molecular mechanisms of repeat expansions have, therefore, attracted a very broad attention.

Our lab got interested in this problem after we discovered, using two-dimensional electrophoretic analysis of the replication intermediates, that replication fork stalled within expandable repeats in vivo. This repeat-mediated fork stalling was observed in bacterial, yeast and mammalian cells. There was a good agreement between the repeat lengths, causing replication blockage in our systems, and their expansion thresholds in human pedigrees. Altogether, these results led us to hypothesize that abnormal replication of expandable repeats could be the cause of their instability.

Subsequently, we have developed a yeast experimental system, which allowed us to analyze large-scale repeat expansions similar to that observed in human pedigrees. This system uncovered several principal features of the repeat expansion process. First, the rate of repeat expansions increased exponentially with their lengths. Second, the median expansion step appeared to correspond to the median size of an Okazaki fragment. Third, vast majority of genes involved in repeat expansions, which came out from unbiased genetic screens, encode protein components of the replication fork. Finally, while every repeat studied so far has a propensity to expand, expansion rates are much higher for structure-prone DNA sequences.

Figure 1

Figure 1. Proposed mechanism for large-scale repeat expansions. Complementary strands of an expandable repeat are shown in red and green. Blue hexameric ring depict the replicative DNA helicase. Purple star designates fork-pausing complex. Yellow circles symbolize leading and lagging DNA polymerases. Gray square shows template switching protein(s).

Based on these observations, as well as data from many other labs, we proposed a model for the large-scale repeat expansions based on the template-switching during DNA replication. It hypothesizes that during replication of a repetitive DNA run (Fig 1A), leading strand DNA polymerase can accidentally (~10–5 per replication) switch its template to continue DNA synthesis along the nascent lagging strand (Fig 1B). Notably, in a long repetitive run, each sequence in the nascent lagging strand sequence is repeated multiple times in the leading strand template. This could make the template switch more feasible, compared to the unique DNA sequences, as an unwound portion of the repetitive leading strand can pair with multiple points along the repetitive lagging strand. After reaching the end of the Okazaki fragment (Fig 1C), the polymerase has to switch back to its primary leading strand template in order for replication to continue. This switch results in the appearance of an expanded repetitive run within the leading DNA strand (Fig 1D).

We are currently continuing to substantiate this model in a yeast experimental system, as well as are investigating its applicability to mammalian cells. In the long run, yeast and mammalian experimental systems for repeat expansions could help searching for drugs that affect the rates of expansions or contractions. These drugs would be invaluable for treatment of the debilitating disorders, caused by expandable repeats.