Timeless tunes

Abstract

Comment on: Leman AR, et al. Cell Cycle 2012; 11:2337-47.

This article refers to:

Timeless preserves telomere length by promoting efficient DNA replication through human telomeres

DNA replication is at the heart of the inheritance of genetic material. A single replication fork can progress through hundreds of kilobases of DNA, melting parental double-stranded DNA and leaving newly synthesized strands in its wake. A beautiful illustration showing how the replication machinery accomplishes this complex task is one of the triumphs of molecular biology. However, it is known that DNA replication is not always as processive as the textbooks suggest. Specifically, the rate of fork progression varies depending on the regions being replicated, and the replication fork even stalls in some circumstances, during replication of heterochromatin or damaged DNA, for example. A stalled replication fork has two fates. It may restart DNA replication, or it may collapse after prolonged stalling. A collapsed replication fork is particularly dangerous for the genome, because the DNA intermediate left by the collapsed fork may form a double-stranded break, a highly mutagenic lesion that can undergo illegitimate recombination. To circumvent replication fork collapse, cells are equipped with specialized proteins that stabilize the stalled replication fork. Timeless and Tipin are highly conserved in eukaryotes. from yeast to humans, and form a complex to protect stalled replication forks.

In a paper published in Cell Cycle, Noguchi and his group investigated how Timeless plays a role in telomere replication in human cells.¹ Telomeres consist of tandem arrays of short repetitive DNA (TTAGGG/CCCTAA in mammals) at the ends of chromosomes and numerous associated proteins. Telomeres are essential for the stable maintenance of genomic DNA, because they protect the DNA termini from undergoing accidental recombination and exonuclease attack. Dysfunctional telomeres lead to genetic instability that eventually results in senescence and cancer development. Because of the heterochromatic nature of telomeres, it has been recognized that telomere DNA is one of the genomic regions that impede replication fork progression. Indeed, in vitro DNA replication experiments using SV40 DNA, and cell extracts demonstrated that telomere DNA is replicated less efficiently and incurs more fork stalling than non-telomeric DNA.² Moreover, overexpression of telomere-DNA binding protein TRF1 in HeLa cells led to an accumulation of replicating telomeres, consistent with a slower replication rate of telomeres under those circumstance. Furthermore, experiments using TRF1-deleted murine cells showed that TRF1 is essential for efficient telomere DNA replication.³ Collectively, these results confirm that the telomere is a difficult-to-replicate region.

There is an apparent contradiction between two earlier studies, however, with TRF1 described as an anti-replication protein in one report² and a pro-replication protein in the other.³ One potential explanation for the inconsistency might be that TRF1 requires other protein(s) to perform its pro-replication function, and the second factor was missing in the TRF1-overexpression experiments. Noguchi and his colleagues investigated this possibility by testing whether Timeless is required for proficient telomere DNA replication.¹ They found that Timeless-knockdown cells displayed telomere length shortening and an increased frequency of dysfunctional telomeres. In vitro replication assays of SV40 DNA revealed that Timeless-depleted extracts supported non-telomere replication proficiently, while telomere replication was inefficient. They then demonstrated that addition of recombinant TRF1 to the replication system slowed telomere replication. Importantly, Timeless depletion and TRF1 addition did not produce additive effects on telomere replication, suggesting that Timeless and TRF1 function in the same pathway. These results suggest a model as described in Figure 1. A replication fork frequently stalls at telomeres because of the molecularly crowded nature of telomeric chromatin. Timeless presumably encounters TRF1 at telomeres and protects the stalled fork from undergoing collapse. In the absence of Timeless, the stalled forks easily collapse, leading to an abrupt shortening of telomeres. Several questions remain to be answered. Given that Timeless moves along the genomic DNA as a component of the replication machinery,⁴ it will be particularly interesting to see how Timeless (or the replication machinery) interacts with telomeric chromatin. In such studies, a dynamic transaction between the regional chromatin at telomeres and the replication machinery may be revealed.

Figure 1. Hard life at telomeres. (A) Mammalian telomeres consist of repetitive DNA that potentially forms higher-ordered structures [G-quartet(G4)-DNA] and numerous proteins, including telomere DNA-binding protein TRF1. (B) Replication fork is frequently stalled at telomeres. Overexpressed TRF1 slows down fork progression at the telomere, while endogenous TRF1 together with Timeless protein facilitates it. Timeless protects the stalled replication fork from collapse. (C) Telomeres are unique in that the most distal replication fork is not coupled with another fork progressing inversely. (D) Prolonged fork stalling may lead to the formation of a DNA double-strand break. Because of the lack of another fork compensating the telomere replication (C), the break immediately results in the abrupt single-step shortening of telomere DNAs.