DNA REPLICATION (2/3) - ELONGATION

At the replication fork, DNA replication enzymes assemble to form the replisome. These enzymes include DNA helicase, DNA polymerases, DNA clamps, RPAs, topoisomerase, DNA ligase, and primase.
Helicase, when activated, promotes DNA melting so one helicase is wrapped around each leading strand of the divergent replication forks. DNA polymerase binds to DNA helicase and the replisome assembles once the DNA strands separate.
DNA polymerases are enzymes that synthesize DNA molecules from individual deoxyribonucleotides. 5 DNA polymerases have major roles in DNA replication: pol α, pol β, pol γ, pol δ, and pol ε. pol α, pol δ, and pol ε are involved in the synthesis of DNA from a template, termed strand elongation.
DNA polymerases have several limitations. They can only synthesize DNA in one direction, 5’ to 3’, adding nucleotides to a previous 3’ hydroxyl group. They also cannot initiate the synthesis of new strands, but can only extend DNA or RNA segments which are paired with a template strand. Therefore, DNA polymerase can only begin DNA synthesis once a primer, a short RNA sequence which is complementary to the template sequence, is added by a primase. Because the primer has a free 3’ hydroxyl group, it can be elongated by DNA polymerase.
Note that DNA strands are antiparallel: the leading strand runs in the 3’ to 5’ direction, but the lagging strand runs 5’ to 3’. But DNA polymerase operates only in the 5’ to 3’ direction. It has no trouble synthesizing DNA on the leading strand, whose complementary strand runs 5’ to 3’. In contrast, the lagging strand is much harder to deal with. It runs 5’ to 3’, so its complementary strand needs to be synthesized 3’ to 5’. Instead of being continuously elongated from one RNA primer, this strand requires multiple RNA primers to be put in by primase. DNA polymerase then comes in to synthesize short DNA fragments called “Okazaki fragments” in between these primers.
pol α, pol δ, and pol ε play different roles, in part because they have different processivity (how many times they can catalyze a reaction without releasing their substrate). Higher processivity means faster DNA synthesis.
Pol α has low processivity and it is poorly suited for efficient synthesis of the nascent DNA strands. It plays a more limited role, initiating DNA replication at origins of replication for both the leading and lagging strands, and for synthesis of Okazaki fragments on the lagging strand. Pol α forms a complex with primase. Primase lays down an RNA primer, which is limited to ~10 nucleotides. Pol α then elongates the RNA primer with ~20 deoxyribonucleotides before handing the job off to another DNA polymerase.
Pol δ has high processivity because it binds to a DNA clamp protein, a sliding clamp that prevents DNA polymerase from dissociating from the DNA parent strand. In eukaryotes, this DNA clamp protein is PCNA. Together, pol δ and PCNA form a holoenzyme. Pol ε has high processivity independently of PCNA.
It is thought that pol δ primarily replicates the lagging strand, and that it removes primers; however, it plays a role in replicating the leading strand as well. Pol ε is thought to continuously synthesize the leading strand and to repair DNA during replication.
How do the RNA primers between Okazaki fragments get removed? Pol δ extends pol α primers until it reaches the 5’ end of the Okazaki fragment. There, it encounters an RNA primer. In order to remove the RNA primer, the pol δ must work with flap endonuclease I, or FEN1. First, the pol δ continues DNA synthesis through limited displacement of the RNA primer. This produces a “5’ flap” in the RNA primer. FEN1 then cuts this flap, and the process repeats again. Through iterative pol δ strand displacement and FEN1 cleavage, the RNA primer is removed. This iterative process is called “nick translation”.
This process has slight complications. The 5’ flap needs to be kept short for FEN1 to retain access to it. To prevent a potential scenario in which pol δ just bulldozes through the entire RNA primer before FEN1 has a chance to act, pol δ has developed an alternative action, called “idling”. Pol δ has a 3’ exonuclease, and it stays at the end of the RNA primer, waiting for FEN1, by using the 3’ exonuclease to cut off the last nucleotide it inserted, then putting it back, then cutting it off, and so on. Eventually, the entire RNA primer gets removed this way, and then DNA ligase can come in and fix the nicks in the nascent DNA strand by forming the missing phosphodiester bonds. This removal of RNA primers, elongation of Okazaki fragments, and ligation is called the maturation of Okazaki fragments.
Now you can see why the lagging strand is synthesized much more slowly than the leading strand. So why doesn’t the leading strand outpace the lagging strand, leaving lots of single stranded DNA exposed on the lagging strand? Well, the primase basically acts as a stop sign, briefly halting the progression of the replication fork.

Видео DNA REPLICATION (2/3) - ELONGATION канала Neural Academy