MutL ATP-dependent conformational changes and the interaction with MutH during DNA mismatch repair processes

Abstract

Robin Holliday first proposed mismatch repair (MMR) in Fungi (Holliday 1964, Modrich 2016), then Wagner and Meselson demonstrated that mismatch repair reaction exists in E. coli cell (Jones, Wagner, et al. 1987, Modrich 2016). Mismatch repair promotes genetic stability, inhibits recombination between non-identical DNA sequences and participating in responses to DNA damage (Harfe and Jinks-Robertson 2000). Deficiency of MMR in humans has been implicated in over 90 % of HPNCC (hereditary nonpolyposis colorectal cancers) (Peltomaki 2005). Lesion detection and removal are the two critical steps of MMR (Yang 2007), MutL bridges this mismatch recognition by MutS and generation a nick at GATC site by MutH endonuclease activity in E.coli MMR processes.Both, bacterial homo-dimeric and eukaryotic hetero-dimeric MutL proteins belong to the GHKL ATPase/kinase superfamily and comprise an N-terminal ATPase domain and C-terminal dimerization regions. All GHKL proteins show substantial ATPase cycle-dependent conformational changes, including ATP binding coupled N-terminal domain dimerization. Interestingly, the ATPase domain of human PMS2 (a subunit of the MutL hetero-dimer) can bind and hydrolyze ATP without dimerization (Iino, Hikima, et al. 2015). The monomeric ATP-bound state of the domain has been thought to be characteristic of hetero-dimeric GHKL proteins (Iino, Hikima, et al. 2015). Hydrolysis of ATP by MutL has been shown to be relatively slow with a kcat of 0.4 min-1 compared with rates between 0.6- 6 min-1 for most ATPase. The ATPase activity of LN40, an N-terminal fragment of MutL, with a kcat of < 0.05 min-1 is almost 10-fold slower than that of the intact MutL (Ban and Yang 1998, Ban, Junop, et al. 1999, Junop, Yang, et al. 2003).Recent publications focused mainly on MutS conformational changes, the complex formation between MutS and MutL (Groothuizen, Winkler, et al. 2015, Qiu, Sakato, et al. 2015, Liu, Hanne, et al. 2016). In contrast, much less in known about conformational changes in MutL induced by ATP and its modulation by the interaction with MutS and G: T mismatch-containing DNA as well as the interaction between MutL and MutH. Studies using atomic force microscopy of the eukaryotic MutL homolog MutLalpha suggested that MutL adopts at least four distinct conformations called extended , one-arm , semi-condensed and condensed , and high ATP drives MutLalpha in to condensed state (Sacho, Kadyrov, et al. 2008). Extended and condensed structures in E. coli MutL ATPase cycle were observed as well about 20 years ago (Ban and Yang 1998, Ban and Yang 1999). To study conformational changes in MutL using Förster resonance energy transfer (FRET), a method for site-specific labeling of MutL and the formation of MutL hetero-dimers with a donor dye label in one domain and an acceptor dye label in the other domain was developed. Heterodimers were generated by a so far unrecognized subunit exchange reaction of the MutL homodimers. Subunit exchanges could be followed in real-time between AF488 (G, donor) and AF594 (R, acceptor) labeled MutL-variants by FRET (GR FRET), e.g., MutL452G and MutL297R hetero-dimer formation in Chapter 1. Analytical ultra-centrifugation (AUC) and single-molecule (sm) FRET confirmed that MutL forms stable (down to 100 pM) yet dynamic (kexchange = 0.11 min-1) dimers.