Modular ssDNA binding and inhibition of telomerase activity by designer PPR proteins

Abstract

DNA is typically found as a double helix, however it must be separated into single strands during all phases of DNA metabolism; including transcription, replication, recombination and repair. Although recent breakthroughs have enabled the design of modular RNA- and double-stranded DNA-binding proteins, there are currently no tools available to manipulate single-stranded DNA (ssDNA). Here we show that artificial pentatricopeptide repeat (PPR) proteins can be programmed for sequence-specific ssDNA binding. Interactions occur using the same code and specificity as for RNA binding. We solve the structures of DNA-bound and apo proteins revealing the basis for ssDNA binding and how hydrogen bond rearrangements enable the PPR structure to envelope its ssDNA target. Finally, we show that engineered PPRs can be designed to bind telomeric ssDNA and can block telomerase activity. The modular mode of ssDNA binding by PPR proteins provides tools to target ssDNA and to understand its importance in cells.

Targeting telomerase for cancer therapy

“…Recent research has provided proof of concept that synthetic RNA-binding pentatricopeptide repeat protein (PPR) mimics of POT1 can bind to telomeric single-stranded DNA (ssDNA) and inhibit telomerase [ 119 ]. The shelterin subunit POT1 binds telomeric ssDNA, thereby antagonizing RPA accumulation and activation of the ATR DDR checkpoint.…”

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery

“…S6 and table S2). Only KRIPP2 and KRIPP10 are large enough to form extended superhelical spiral structures, and only KRIPP2 wraps around single-stranded RNA in a prototypical manner (22,23), specifically recognizing the 5′-end region of the 9S rRNA and cradling the rRNA with its positively charged interaction surface (fig. S9).…”

Manipulating and elucidating mitochondrial gene expression with engineered proteins

“…Furthermore, there are other programable DNA-binding proteins that have yet to be exploited for mitochondrial genome engineering. For example, engineered pentatricopeptide repeat (PPR) proteins can be programmed to recognize specific single-stranded DNA sequences in a modular manner [93], providing potential tools to hijack mtDNA replication and recombination.…”

“…The stability of cPPR proteins has enabled a variety of crystal structures of these proteins to be solved. Unlike PUF proteins which do not change conformation significantly upon RNA binding and associate with predominantly the Watson-Crick face of the RNA, PPR proteins undergo massive compaction when they bind RNA and wrap around their targets extensively [93,125]. These differences could enable distinct applications in mtRNA manipulation, particularly in masking RNAs from the actions of enzymes that normally target them [93].…”

“…Unlike PUF proteins which do not change conformation significantly upon RNA binding and associate with predominantly the Watson-Crick face of the RNA, PPR proteins undergo massive compaction when they bind RNA and wrap around their targets extensively [93,125]. These differences could enable distinct applications in mtRNA manipulation, particularly in masking RNAs from the actions of enzymes that normally target them [93]. Furthermore, in contrast to naturally occurring PUF proteins, natural PPR proteins characterized in a variety of systems to date have roles in organelles and have often been observed to contain many other domains with diverse roles in RNA metabolism, such as RNA cleavage, modification, and control of translation [120].…”