The TIMELESS effort for timely DNA replication and protection

Abstract

Accurate replication of the genome is fundamental to cellular survival and tumor prevention. The DNA replication fork is vulnerable to DNA lesions and damages that impair replisome progression, and improper control over DNA replication stress inevitably causes fork stalling and collapse, a major source of genome instability that fuels tumorigenesis. The integrity of the DNA replication fork is maintained by the fork protection complex (FPC), in which TIMELESS (TIM) constitutes a key scaffold that couples the CMG helicase and replicative polymerase activities, in conjunction with its interaction with other proteins associated with the replication machinery. Loss of TIM or the FPC in general results in impaired fork progression, elevated fork stalling and breakage, and a defect in replication checkpoint activation, thus underscoring its pivotal role in protecting the integrity of both active and stalled replication forks. TIM is upregulated in multiple cancers, which may represent a replication vulnerability of cancer cells that could be exploited for new therapies. Here, we discuss recent advances on our understanding of the multifaceted roles of TIM in DNA replication and stalled fork protection, and how its complex functions are engaged in collaboration with other genome surveillance and maintenance factors.

The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

“…By implementing both a degron that rapidly degrades endogenous TIM and an inducible polypeptide that disrupts the TIM–PARP1 interaction, we have determined that the TIM–PARP1 complex is an essential element of the DNA replication machinery necessary for efficient DNA replication fork elongation. As a key constituent of the FPC, TIM, together with its obligate heterodimer TIPIN, tethers the CMG helicase and replicative polymerase activities to maintain the integrity of the replisome ( 2 , 33 , 34 ). Positioning of TIM on dsDNA at the leading edge of CMG stabilizes the replisome and facilitates the separation of leading and lagging strands ( 3 , 4 ).…”

“…DNA replication is achieved by the coordinated action of the replisome that couples the unwinding activity of the CDC45/MCM2-7/GINS (CMG) helicase complex and incorporation of nucleotides complementary to a DNA template by replicative polymerases ( 1 ). The fork protection complex (FPC), which consists of TIMELESS (TIM), TIPIN, CLASPIN and AND-1 in mammalian cells, plays a key role in maintaining the integrity of the replisome and coupling its activity, thus enabling efficient replication fork progression ( 2 ). The TIM-TIPIN heterodimer (Tof1-Csm3 in Saccharomyces cerevisiae ) grips double-stranded DNA (dsDNA) in front of the CMG to stabilize the association of the replisome to DNA and facilitate strand separation ( 3 , 4 ).…”

The Adaptive Mechanisms and Checkpoint Responses to a Stressed DNA Replication Fork

“…The activities of DNA unwinding and synthesis in the replisome need to be tightly coupled; otherwise, uncoupling of replisome activity results in a large stretch of ssDNA accumulation that can lead to fork breakage and collapse, which is considered a major source of chromosome aberrations and instability. The architecture of the replication fork is primarily maintained by the fork protection complex (FPC), which physically interacts with the replication machinery and tethers the CMG helicase and replicative polymerase activities to prevent uncoupling of DNA replication, and thus limit ssDNA exposure [ 28 ]. The FPC is composed of a heterodimeric complex of TIMELESS (TIM) and TIPIN (Tof1 and Csm3 in S. cerevisiae ; Swi1 and Swi3 in S. pombe ), as well as CLASPIN/Mrc1 and AND-1/Ctf4 [ 29 ] ( Figure 2 ).…”

Phosphorylation inhibits intramolecular interactions, DNA-binding and protein interactions of Claspin through disordered/ structured conformation transition

“…At the eukaryotic DNA replication fork, the CMG (Cdc45-Mcm-GINS) complex serves as a replicative helicase, in which the Mcm hexameric complex plays a major role as an active unwinding enzyme. At the progressing replication fork, other accessory factors including Claspin, Tim, Tipin and And-1 play crucial role in facilitating the fork movement and stabilizing replication forks upon replication stress, generating a large replisome progression complex (RPC) 1,2,3,4,5,6 .…”