Peter Cherepanov: Projects

Retroviral DNA integration

Retroviral replication requires stable insertion of the viral genetic material into cellular genome. The catalytic events associated with this process are carried out by the virus-derived enzyme integrase (IN).

Retroviral INs are structurally and mechanistically related to a diverse group of enzymes including bacterial and eukaryotic transposases, V(D)J recombinase RAG1/2, ribonuclease H, and the catalytic subunit of the RNA-induced silencing complex (RISC).

This superfamily is distinguished by the characteristic structural fold, organization of the active site and metal-dependent catalysis. As pathogens, viruses present an obvious societal burden and, being of the three essential retroviral enzymes, IN is an important target for anti-HIV/AIDS drug development. On the flipside, the unique ability of retroviruses to efficiently integrate their genetic material into host cell chromosomal DNA makes them ideal genome manipulation tools.

In principle, a single application of a retroviral vector may be sufficient to correct a genetic deficiency, make cells resistant to an infection or help the immune system to fight off cancer. Yet, uncontrolled integration of retroviral vectors poses an inherent risk of insertional mutagenesis and, consequently, serious side effects.

Over the years we determined a collection of crystal structures, which illustrated the mechanism of the retroviral integration process and revealed the mode of action of clinical HIV-1 IN inhibitors, such as Raltegravir. We also determined crystal structures of Transportin 3 (Tnpo3), the B-karyopherin involved in the early steps of HIV-1 replication in addition to its role in nuclear import of cellular Ser/Arg-rich splicing factors.

The brunt of our current efforts is currently directed towards understanding the rules of engagement between the retroviral integration machinery and the cellular environment. We hope that our research will yield the keys to developing safer retroviral vectors as well as novel concepts for anti-HIV/AIDS therapies. The two major projects aim to elucidate the structural and functional features of the interface between integrase and chromatin and the identification and the roles of host cell factors during integration.

Figure 1

Figure 1. (A) Crystal structure of the intasome after engaging target (genomic) DNA in the strand transfer complex. (B) IN active site before (top) and after binding Raltegravir (RAL). Active site residues comprising the active site DDE motif of IN are indicated. (C) Crystal structure of TNPO3 in complex with its cellular cargo ASF/SF2. This panel is adapted from Maertens et al., Proc Natl Acad Sci USA, 2014, 111:2728–33.

Factors involved in initiation of eukaryotic DNA replication

Eukaryotic replisome assembly and initiation of the DNA synthesis at individual origins require actions of many proteins and critically depend on activities of S-phase cyclin-dependent kinases (S-CDKs) and Cdc7-Dbf4. Both types of heterodimeric kinases are regulated by their respective activating subunits (cyclins and Dbf4), which oscillate during the cell cycle.

The most characterised role of Cdc7-Dbf4 is phosphorylation of the MCM2-7 complex, required to trigger its DNA helicase activity. Due to its essential role in DNA replication, Cdc7-Dbf4 has been put forward as a target for the development of anti-cancer drugs.

We use x-ray crystallography and complementary approaches to gain structural insights in the initiation of DNA replication. Recently we determined the first crystal of Cdc7-Dbf4 kinase, which elucidated the basis for binding to and activation of Cdc7 by Dbf4 and provided a framework for design of more potent and specific Cdc7 inhibitors.

We are now focusing on the mechanisms of regulation of Cdc7 activity and other structural aspects of initiation of eukaryotic DNA replication.

Figure 2

Figure 2. (A) Crystal structure of the Cdc7-Dbf4 heterodimer. Cdc7 is colored in green (canonical part of the N-lobe structure), purple (canonical C-lobe structures) and yellow (structures unique to Cdc7) and Dbf4 in orange. (B) Views on the Cdc7 active site engaged with inhibitors PHA767491 (top) or XL413 (bottom). Cdc7 surface is colored according to conservation, with least conserved atoms are shown in red and those most conserved in gray. This figure is adapted from Hughes et al., Nat Struct Mol Biol, 2012, 19:1101–7.

 

 

Peter Cherepanov

peter.cherepanov@crick.ac.uk
+44 (0)20 379 61798

  • Qualifications and history
  • 2000 PhD Rega Institute for Medical Research, 2000 Postdoctoral fellow, Rega Institute for Medical Research, Belgium
  • 2003 Postdoctoral fellow, Department of Pathology, Harvard Medical School, Boston, USA
  • 2005 Senior Lecturer, Faculty of Medicine,2009 Reader in Virology, Faculty of Medicine, Imperial College London, UK
  • 2011 Established lab at the London Research Institute, Cancer Research UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK