By promoting replication through DNA lesions, translesion synthesis (TLS) DNA polymerases (Pols) play a
critical role in preventing chromosomal instability and protecting against tumorigenesis. Unlike replicative Pols,
TLS Pols have less constrained active sties and they lack proofreading 3'¿5' exonuclease activity.
Consequently, purified TLS Pols synthesize DNA opposite DNA lesions with an extremely low fidelity. Despite
this, TLS operates in a predominantly error-free manner in normal human cells (not derived from cancers), The
overall objective in this project is to identify the cellular processes and mechanisms by which high fidelity is
imposed upon TLS by the intrinsically highly error-prone Y-family Pols. Using a combination of genetic, cellular,
biochemical, and structural approaches, we will address the following questions: (1) Do the Y-family Pols
associate with other protein factors in a multiprotein ensemble and do these proteins have activities that elevate
the fidelity of the TLS Pol? (2) What is the protein composition of the entire Y-family Pol ensemble for error-free
TLS in human cells? (3) How is the fidelity of TLS modulated by the components of the multiprotein ensemble?
(4) What are the molecular underpinnings of action mechanisms via which components of the multiprotein
ensemble impose high fidelity on Y-family Pols? To pursue these questions, we have identified a number of
protein factors that function in TLS specifically in conjunction with Y-family Pols; included among these proteins
are WRN which possesses DNA helicase and 3'¿5' exonuclease activities, and WRNIP1 which has a DNA
dependent ATPase activity. How these activities contribute to the fidelity of TLS by Y-family Pols opposite
different types of DNA lesions will be analyzed in extensive mutational studies that include genome wide
sequencing. Using proximity labeling in which TurboID is fused to Pol¿, we will determine whether there are
additional proteins that function in TLS in conjunction with Y-family Pols and whether activities in these proteins
affect the fidelity of TLS by these Pols. In biochemical studies with the purified multiprotein ensemble of Pol¿ or
Pol¿, we will ascertain the roles of WRN 3'¿5' exonuclease, WRN and WRNIP1 ATPase, and of any other newly
identified activities in the high fidelity of TLS by these Pols opposite different types of DNA lesions. From cryo-
EM studies with the purified multiprotein ensemble of Pol¿ or Pol¿, we will determine mechanistically how the
components of the multiprotein ensemble modulate the fidelity of these Y-family Pols opposite DNA lesions.
Cumulatively, these studies will identify the components of the multiprotein Y-family TLS replicases which
carry out high fidelity TLS in human cells. They will reveal the mechanisms by which the various components
constrain Y-family Pols' active sites to restrain nucleotide (nt) misincorporation and how WRN's 3'¿5'
exonuclease activity is coordinated with the TLS Pol for the removal of misinserted nt. These studies will be
paradigm shifting and will open new vistas of research into the mechanistic details of TLS Pols' fidelity and they
will give impetus to further elaboration of the roles of TLS Pols in genome integrity.