This second end-replication problem means that both strands of DNA will shorten with each division. The replisome does not generate Okazaki fragments on the 3' overhang it actually stops lagging-strand synthesis long before the leading strand reaches the 5' end. The results of Yeeles' in vitro replication experiments were very clear. Could the replisome use a 3' overhang to make the last Okazaki fragment, as was proposed? Yeeles agreed that it would be good to take a close look at how the replisome behaves at the end of a linear DNA template. Yeeles, a biochemist who studies DNA replication at the Laboratory of Molecular Biology in Cambridge (the same lab where Watson and Crick worked on the structure of the DNA double helix). As his results seemed very solid to me, we needed to revisit the model."ĭe Lange contacted Joseph T. "At that point, Hiro and I realized that either his results were not right or the model was wrong. "The results just didn't fit with the model for telomere replication," de Lange says. Takai's work suggested that the end-replication problem was twice as serious as previously thought, impacting both strands of DNA. This new data revealed something unexpected: not only was the leading strand in need of help-he found evidence that the end of the lagging strand could also not be synthesized by the replisome. He and others had previously shown that CST–Polα-primase can replenish CCCTAA repeats at telomeres that had been attacked by DNA-degrading enzymes known as nucleases. Until Takai made a surprising discovery while working on cells that lacked molecular machinery called the CST–Polα-primase complex. Credit: Credit Sarah CaiĪs descriptions of biological processes go, this model looked watertight. CST–Polα/primase, the enzyme that solves the newly discovered end-replication problem. "The DNA replication machinery cannot not fully duplicate the end of a linear DNA, much the same way that you can't paint the floor under your feet," says Hiro Takai, senior staff scientist in the de Lange lab and lead author on the paper. It could start the last Okazaki fragment somewhere along the 3' overhang. As for the lagging-strand, DNA synthesis should not have a problem. Telomerase solves this problem by adding single-stranded TTAGGG repeats to the telomere end. The end-replication problem arises because leading strand synthesis fails to reproduce the last part of the telomere, leaving a blunt leading-end telomere without it characteristic and crucial 3' overhang. When copying the telomere, leading-strand DNA replication should copy the CCCTAA repeats to generate the TTAGGG repeat strand, while lagging-strand synthesis should do the opposite, making new CCCTAA repeats. The process is fairly direct until the ends of the chromosomes. But the other strand is synthesized in short backward steps from many fragments (Okazaki fragments) that are later stitched together. The replisome copies the 3' to 5' strand without interruption, a process referred to as leading-strand synthesis. When DNA is replicated, the two strands are separated by the replication machinery, also called the replisome. Since the description of the DNA double helix, it is known that DNA has two complementary strands running in opposite directions-one from 5' to 3' the other from 3' to 5'. "It turns out we had missed half the problem." The leading-strand problem "For many decades we thought we knew what the end-replication problem was and how it was solved by telomerase," says de Lange. Further, telomerase is only part of the solution-cells also use the CST–Polα-primase complex, which has been extensively studied in de Lange's laboratory. Now, research published in Nature suggests that there are two end-replication problems, not one. "Case closed, everybody thought," says Rockefeller's Titia de Lange.
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