Malgorzata Figiel

I wish to thank all the co-authors from @iimcb.bsky.social and from @hauryliuk.bsky.social lab at @lunduniversity.bsky.social !

E. coli DRT7 provides defense against a very broad range of phages and uses a direct defense mechanism. The escape mutants of phage lambda and Bas1 that overcame this protection carried mutations in genes encoding a DNA mimic and a putative phosphoesterase, respectively.

Functional tests confirm that UG10 RTs synthesize double-stranded DNA, but in DRT7 the tip of the arch domain comes between the priming motif and the primase domain. We envisage the conformational changes that could allow the template strand to reach the primase active site.

Cryo-EM reconstruction for an RT-dead variant of UG10S revealed a rearrangement of the two-helix motif that places the priming tyrosine at the RT active site. We predict this to be the conformation required for the attachment of the first nucleotide of the nascent product.

The covalent link between the co-purified DNA and the (P)rotein is visualized in the structure and confirmed in a functional assay, where the DNA product was (L)abeled with a fluorescent nucleotide and extended in a (R)eaction with dTTP.

We determined cryo-EM structures of two representatives of UG10 RT family – a “large” system from E. coli, known as DRT7, and the RT-part of a “small” system from S. salivarius; both with a DNA product. A notable difference: the relative positions of the subdomains.

Excited to share our preprint exploring the structural, biochemical and in vivo properties of DRT7/UG10 reverse transcriptases which produce dsDNA using their primase and polymerase activities and provide broad-spectrum antiphage defense! www.biorxiv.org/content/10.6...

I wish to thank all the coauthors of this study and our colleagues from Nowotny lab. We are also grateful to @iimcb.bsky.social, @ncn.gov.pl and Solaris National Synchrotron Radiation Centre for making this project possible!

Finally, we deliver the first complete cryo-EM structure of a two-component recombinase composed of ICP8 annealase and UL12 alkaline nuclease. The complex was reconstituted using a model DNA with a dsDNA and 3′ ssDNA overhang.

We are also presenting a cryo-EM reconstruction of a double filament of ICP8 at 3.2 Å resolution. We visualize the interactions between protein subunits within and between the antiparallel strands. DNA used to produce the filaments could not be traced.

A very similar DNA conformation is observed in the crystal structures of ICP8 with 14-mer ssDNA. ICP8 subunits form dimers with a two-fold symmetry, with their DNA-binding sites facing each other and the outward-rotated bases of bound DNAs form pairs in a flexible manner.

We determined a cryo-EM structure of BALF2 bound to ssDNA at 3.2 Å resolution. The visible fragment of DNA is accommodated in a positively charged cleft. In the first four nucleotides, the base edges face the protein, but the next three bases are rotated away from the protein.

I am very excited to share our recent preprint in which we are structurally investigating macromolecular complexes that contain DNA-binding proteins ICP8 of Herpes Simplex Virus 1 and BALF2 of the Epstein-Barr Virus.
biorxiv.org/content/earl...

We are very happy to share the preprint of our latest study on the DNA-binding proteins from HSV-1 and EBV! @iimcb.bsky.social