Becker, Nicole A.

Using DNA ligase‐catalyzed cyclization assays, the in vitro persistence length of DNA has been determined to be about 150 base pairs (bp). In living E. coli bacteria, where DNA loops regulate the lac operon, DNA looping appears to be much more probable than expected from in vitro data. Why? A possible explanation for this paradox is that DNA loops in bacteria are bridged by proteins, reducing the amount of required costly DNA bending. Current in vitro methods to measure DNA flexibility are tedious and artificial, in that they require DNA cyclization, not just DNA looping. No high‐throughput method has been described to measure DNA flexibility for loops involving bridging proteins of different sizes. We are developing a high‐throughput method to measure the probability of DNA looping when bridged by a fusion protein that includes PCV2 and micrococcal nuclease domains. A bead‐tethered 1000‐bp DNA molecule of a known sequence is conjugated to this fusion protein through the PCV2 domain, and looping allows self‐cleavage such that the distribution of cleavage sites revealed by next generation sequencing reflects the energetics of DNA looping. We hypothesize that this distribution will be affected as a function of bridging protein size. Measuring how the size of bridging proteins affects tight DNA loop probability enables improved understanding of genetic switches.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.

Significance Double-stranded DNA is one of the stiffest polymers in biology, resisting both bending and twisting over hundreds of base pairs. However, tightly bent DNA loops are formed by proteins that turn off (repress) genes in bacteria. It has been shown that “architectural” proteins capable of kinking any DNA molecule without sequence preference facilitate this kind of gene repression. The mechanism of this effect is unknown for DNA loops involving the well-known Escherichia coli lac repressor. Here we adapt high-resolution protein-mapping techniques to show that an architectural protein directly binds tightly looped DNA to facilitate gene repression by the lac repressor.