Cell Biology | DNA Replication 🧬

DNA replication is essential for cell replication, occurring primarily in the S phase of the cell cycle. This process produces two identical daughter cells, each with replicated DNA. DNA replication is semi-conservative, meaning each new DNA molecule consists of one old (parental) strand and one new (daughter) strand. Replication always proceeds in a 5' to 3' direction and is bidirectional from the origin of replication, forming replication forks.

Initiation begins at specific regions called origins of replication, which are rich in adenine-thymine (A-T) base pairs because they are easier to break (two hydrogen bonds vs. three in G-C pairs). In eukaryotes, there are multiple origins of replication. The pre-replication protein complex binds to these origins, separating the DNA strands and forming a 'replication bubble.' Single-stranded binding proteins then attach to the separated strands, preventing them from re-annealing and protecting them from nucleases. Helicase enzymes then move to the replication forks, unwinding the DNA in an ATP-dependent manner.

As helicase unwinds DNA, it creates supercoils downstream, which can impede further unwinding. Topoisomerases relieve this tension by cutting and then rejoining the DNA strands. There are different types: Type 1 (eukaryotic) does not require ATP, while Type 2 (eukaryotic) and Type 2 and 4 (prokaryotic, also known as DNA gyrase) require ATP. Clinically, topoisomerases are targets for cancer drugs (e.g., irinotecan, topotecan for Type 1; etoposide, teniposide for Type 2) and bacterial infection treatments (fluoroquinolones for prokaryotic Type 2/4). These drugs work by enhancing the nuclease activity while inhibiting the ligase activity of topoisomerases, leading to DNA fragmentation.

Elongation involves primase laying down short RNA primers, as DNA polymerase III cannot initiate DNA synthesis de novo; it requires a 3' OH group. Primase reads the DNA template from 3' to 5' and synthesizes RNA primers from 5' to 3'. DNA polymerase III then binds to the 3' OH of the RNA primer, reads the DNA template 3' to 5', and synthesizes new DNA 5' to 3'. This occurs continuously on the leading strand (requiring one primer) and discontinuously on the lagging strand, creating Okazaki fragments (multiple primers and DNA stretches).

DNA polymerase III possesses a 3' to 5' exonuclease proofreading activity, correcting mismatched nucleotides. After DNA synthesis, RNA primers must be removed. DNA polymerase I performs this by its 5' to 3' exonuclease activity, removing RNA primers and simultaneously replacing them with DNA nucleotides. It also has a 3' to 5' exonuclease proofreading activity. On the lagging strand, after DNA polymerase I replaces RNA primers with DNA, small gaps remain. DNA ligase then fuses these DNA segments together, creating a continuous strand.

Nucleoside Reverse Transcriptase Inhibitors (NRTIs), used in HIV treatment, are nucleoside analogs that lack a 3' OH group. When incorporated into a growing DNA strand by reverse transcriptase, they terminate the elongation process, halting viral replication. This highlights how understanding DNA replication mechanisms can inform therapeutic strategies.

DNA replication terminates when helicases from opposing replication forks meet and their activity ceases. DNA polymerases then detach. A unique challenge arises at the ends of linear chromosomes, called telomeres. Due to the inability of DNA polymerase to synthesize DNA at the very end of the lagging strand template, telomeres shorten with each replication cycle. This shortening prevents gene loss since telomeres do not code for RNA and protect the integrity of coding regions. The Hayflick limit describes the maximum number of cell divisions before critical genes are affected by telomere shortening.

Telomerase, a ribonucleoprotein, addresses telomere shortening in rapidly dividing cells (e.g., stem cells). It extends the telomeres by using its internal RNA template to synthesize complementary DNA sequences (a process called reverse transcription). This mechanism prevents significant telomere shortening and associated gene loss. Cancer cells often upregulate telomerase activity, allowing them to replicate indefinitely, a key characteristic of neoplasia.