Half a century in the past, scientists Jim Watson and Alexey Olovnikov independently realized that there was an issue with how our DNA will get copied. A quirk of linear DNA replication dictated that telomeres that shield the ends of chromosomes ought to have been rising shorter with every spherical of replication, a phenomenon generally known as the end-replication downside.
However an answer was forthcoming: Liz Blackburn and Carol Greider found telomerase, an enzyme that provides the telomeric repeats to the ends of chromosomes. “Case closed, all people thought,” says Rockefeller’s Titia de Lange.
Now, new analysis revealed in Nature means that there are two end-replication issues, not one. Additional, telomerase is just a part of the answer — cells additionally use the CST-Polα-primase advanced, which has been extensively studied in de Lange’s laboratory. “For a lot of a long time we thought we knew what the end-replication downside was and the way it was solved by telomerase,” says de Lange. “It seems we had missed half the issue.”
The leading-strand downside
For the reason that description of the DNA double helix, it’s recognized that DNA has two complementary strands operating in reverse instructions — one from 5′ to three’; the opposite from 3′ to five’. When DNA is replicated, the 2 strands are separated by the replication equipment, additionally known as the replisome. The replisome copies the three’ to five’ strand with out interruption, a course of known as leading-strand synthesis. However the different strand is synthesized in brief backward steps from many fragments (Okazaki fragments) which are later stitched collectively.
The method is pretty direct till the ends of the chromosomes. When copying the telomere, leading-strand DNA replication ought to copy the CCCTAA repeats to generate the TTAGGG repeat strand, whereas lagging-strand synthesis ought to do the other, making new CCCTAA repeats. The tip-replication downside arises as a result of main strand synthesis fails to breed the final a part of the telomere, leaving a blunt leading-end telomere with out it attribute and essential 3′ overhang. Telomerase solves this downside by including single-stranded TTAGGG repeats to the telomere finish. As for the lagging-strand, DNA synthesis shouldn’t have an issue. It might begin the final Okazaki fragment someplace alongside the three’ overhang.
“The DNA replication equipment can’t not absolutely duplicate the tip of a linear DNA, a lot the identical method you can’t paint the ground underneath your ft” says Hiro Takai, senior workers scientist within the de Lange lab and lead writer on the paper.
The lagging-strand downside
As descriptions of organic processes go, this mannequin appeared watertight. Till Takai made a shocking discovery whereas engaged on cells that lacked molecular equipment known as the CST-Polα-primase advanced. He and others had beforehand proven that CST-Polα-primase can replenish CCCTAA repeats at telomeres that had been attacked by DNA-degrading enzymes generally known as nucleases. This new knowledge revealed one thing surprising: not solely was the main strand in want of assist — he discovered proof that the tip of the lagging strand might additionally not be synthesized by the replisome.
Takai’s work instructed that the end-replication downside was twice as severe as beforehand thought, impacting each strands of DNA. “The outcomes simply did not match with the mannequin for telomere replication,” de Lange says. “At that time, Hiro and I spotted that both his outcomes weren’t proper or the mannequin was incorrect. As his outcomes appeared very stable to me, we would have liked to revisit the mannequin.”
De Lange contacted Joseph T. P. Yeeles, a biochemist who research DNA replication on the Laboratory of Molecular Biology in Cambridge (the identical lab the place Watson and Crick labored on the construction of the DNA double helix). Yeeles agreed that it might be good to take an in depth take a look at how the replisome behaves on the finish of a linear DNA template. May the replisome use a 3′ overhang to make the final Okazaki fragment, as was proposed?
The outcomes of Yeeles’ in vitro replication experiments had been very clear. The replisome doesn’t generate Okazaki fragments on the three’ overhang; it truly stops lagging-strand synthesis lengthy earlier than the main strand reaches the 5′ finish. This second end-replication downside implies that each strands of DNA will shorten with every division. Telomerase was solely stopping this from occurring on the main strand and Hiro’s knowledge instructed that CST-Polα-primase mounted the second end-replication downside, that of the lagging strand.
Takai spent the following 4 years designing new assays to substantiate Yeeles’ findings in vivo. He was in a position to measure how a lot DNA is misplaced as a result of lagging-strand end-replication downside, revealing what number of CCCAAT repeats have to be added by CST-Polα-primase to maintain telomeres intact.
The outcomes change our understanding of telomere biology — requiring revision of the textbooks. However the findings might also have scientific implications. People who inherit mutations in CST-Polα-primase undergo from telomere issues, akin to Coats plus syndrome, which is characterised by an eye fixed dysfunction and abnormalities within the mind, bones, and GI tract. By means of a greater understanding of how we keep our telomeres, strides might sooner or later be made in addressing these devastating issues.
