Biology subjects

Furman, C. M.

Publications and source records attributed to Furman, C. M..

4 recordsLinked to original sources

bioRxiv2022

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells

Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac. The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

molecular biology↗

bioRxiv2022

Formation of the active Hermes transpososome is driven by asymmetric DNA binding of BED domains

The cut-and-paste Hermes DNA transposase stands out among the transposases that have been biochemically or structurally characterized so far. Many transposases function as dimers, but the Hermes transposase forms a tetramer of dimers to achieve its active form in vivo. Intriguingly, the transposition complex, or transpososome, relies on only one dimer to perform the enzymatic reactions necessary to the mobilization of its transposon. Our investigation combining biochemical and structural approaches shows that the Hermes octamer extensively interacts with its transposon left-end (LE) engaging the BED domains of three Hermes protomers belonging to three dimers. By contrast, the right-end (RE) is entirely deprived of such interaction inside the transpososome. Our work suggests that formation of the Hermes synaptic complex is sequential and relies on the considerable difference of affinity of the transposase towards its transposon ends. Thus, we propose that Hermes dimers multimerize to gather enough BED domains to find the LE among the abundant genomic DNA, facilitating the subsequent interaction with the RE, most likely solely based on recognition of its terminal inverted repeat.

biochemistry↗

bioRxiv2021

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs). While essential for MMR, their exact functions remain elusive. We performed cross-linking mass spectrometry to identify the major interactions within the Mlh1-Pms1 heterodimer and used this information to insert FRB and FKBP dimerization domains into the IDRs of Mlh1 and Pms1. Yeast bearing these constructs were grown with rapamycin to induce dimerization. Strains containing FRB and FKBP domains in the Mlh1 IDR displayed complete MMR defects when grown with rapamycin, but removing rapamycin restored MMR functions. Furthermore, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization disrupted Mlh1-Pms1 binding to DNA, inappropriately activated Mlh1-Pms1, and caused MMR defects in vivo. We conclude that dynamic and coordinated rearrangements of the MLH IDRs regulate how the complex clamps DNA to catalyze MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

genetics↗

bioRxiv2018

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex.

Intrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry, and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutL (Mlh1-Pms1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the ATP hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutL in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.

biochemistry↗