Replication

Replication of DNA

• DNA exists in many forms: single- and double-stranded, linear and circular
• Many DNAs are double-stranded, and the process of replication is complex, with a high degree of fine-tuning to ensure fidelity
• The cell faces three important challenges in replication:
  • Separating the two DNA strands
  • Synthesizing DNA from the 5' to the 3' end
  • Guarding against errors in replication

Semiconservative Replication

• DNA replication involves the separation of the two original strands and the production of two new strands with the original strands as templates
• Each new DNA molecule contains one strand from the original DNA and one newly synthesized strand
• This process is called semiconservative replication

How did scientists figure out that replication is semiconservative?

• Experiments by Matthew Meselson and Franklin Stahl in the late 1950s used E. coli bacteria grown with a heavy isotope of nitrogen (15N)
• The 15N-labeled DNA has a higher density than unlabeled DNA
• The cells were then transferred to a medium with the usual isotope of nitrogen (14N), and the DNA was analyzed using density-gradient centrifugation
• The results confirmed the predictions of semiconservative replication

Direction of Replication

• Replication proceeds bidirectionally from the origin of replication
• New polynucleotide chains are synthesized using each of the exposed strands as a template
• Two replication forks advance in opposite directions from the origin of replication

DNA Polymerase

• All synthesis of nucleotide chains occurs in the 5' to 3' direction
• The last nucleotide added to a growing chain has a 3'-hydroxyl on the sugar
• The incoming nucleotide has a 5'-triphosphate on its sugar
• The 3'-hydroxyl group at the end of the growing chain is a nucleophile that attacks the phosphorus adjacent to the sugar in the nucleotide to be added

Semidiscontinuous DNA Replication

• The two strands of DNA are going in opposite directions during replication
• The problem is solved by different modes of polymerization for the two growing strands
• One newly formed strand (the leading strand) is formed continuously from its 5' end to its 3' end
• The other strand (the lagging strand) is formed semidiscontinuously in small fragments (Okazaki fragments)

Properties of DNA Polymerases

• At least five DNA polymerases are present in E. coli
• The properties of DNA polymerases include:
  • Turnover number (speed of the synthetic reaction)
  • Processivity (the number of nucleotides joined before the enzyme dissociates from the template)
  • Proofreading and repair functions
• DNA polymerase III has the highest turnover number and a huge processivity compared to polymerases I and II

Primase Reaction

• RNA serves as a primer in DNA replication
• The primer activity of RNA was first observed in vivo
• A separate enzyme, called primase, is responsible for copying a short stretch of the DNA template strand to produce the RNA primer sequence
• The primer and the protein molecules at the replication fork constitute the primosome

Synthesis and Linking of New DNA Strands

• The synthesis of two new strands of DNA is begun by DNA polymerase III
• The newly formed DNA is linked to the 3'-hydroxyl of the RNA primer
• Synthesis proceeds from the 5' end to the 3' end on both the leading and the lagging strands
• The RNA primer is removed by polymerase I, using its exonuclease activity
• The primer is replaced by deoxynucleotides, also by DNA polymerase I
• DNA ligase seals the remaining nicks

Architecture of DNA Polymerases and the Replisome

• The various DNA polymerases have a common structure that is often compared to a right hand
• The active site where the polymerase reaction is catalyzed lies in the crevice within the palm domain
• The fingers domain acts in deoxynucleotide recognition and binding
• The thumb is responsible for DNA binding

A Summary of DNA Replication in Prokaryotes

• DNA synthesis is bidirectional
• The direction of DNA synthesis is from the 5' end to the 3' end of the newly formed strand
• One strand (the leading strand) is formed continuously, while the other strand (the lagging strand) is formed discontinuously
• Five DNA polymerases have been found in E. coli
• DNA gyrase introduces a swivel point in advance of the movement of the replication fork
• A helix-destabilizing protein, a helicase, binds at the replication fork and promotes unwinding
• The exposed single-stranded regions of the template DNA are stabilized by a DNA-binding protein
• Primase catalyzes the synthesis of an RNA primer
• The synthesis of new strands is catalyzed by Pol III
• The primer is removed by Pol I, which also replaces the primer with deoxynucleotides
• DNA ligase seals the remaining nicks### DNA Replication and Repair
• DNA replication is an essential process that occurs only once in each cell generation, and it's crucial to ensure the fidelity of the replication process to prevent mutations.
• The process of DNA replication is semiconservative, meaning that each new DNA molecule is composed of one old strand (the template) and one new strand synthesized during replication.

Proofreading and Repair

• Proofreading refers to the removal of incorrect nucleotides immediately after they are added to the growing DNA during the replication process.
• DNA polymerase I has three active sites, including the polymerase activity, proofreading activity, and 5' S 3' repair activity.
• Proofreading improves the fidelity of replication to one error in every 10^9 to 10^10 base pairs.
• The cut-and-patch process catalyzed by polymerase I takes place after polymerase III has produced the new polynucleotide chain.
• Existing DNA can also be repaired by polymerase I using the cut-and-patch method.

Mismatch Repair

• Mismatch repair is a process that recognizes and corrects errors in DNA replication.
• Enzymes recognize incorrectly paired bases, and the area with the mismatch is removed, and DNA polymerases replicate the area again.
• The challenge for the repair system is to know which of the two strands is the correct one.
• Prokaryotes alter their DNA at certain locations by adding methyl groups, which helps the repair system distinguish between the parental and newly synthesized strands.

Nonhomologous DNA End-Joining (NHEJ)

• NHEJ is a repair mechanism that handles double-stranded breaks (DSBs) in DNA.
• A heterodimeric protein called Ku70/80 binds the broken ends of the DNA and recruits several other proteins that repair the damage.
• This repair mechanism proceeds without a template, making it an error-prone mechanism.

Homologous Recombination

• Homologous recombination is a natural process that rearranges genetic information to form new associations.
• It involves the exchange of one DNA sequence with another or the incorporation of a DNA sequence into another.
• The process is critical during meiosis, ensuring the correct segregation of chromosomes.
• Hot spots are areas of a chromosome more likely to show recombination.

Eukaryotic DNA Replication

• Eukaryotic replication is more complicated than prokaryotic replication, with multiple origins of replication, controlled timing, and more proteins involved.
• Replication begins at multiple origins of replication, also called replicators, and occurs in the S phase of the cell cycle.
• The process is tied to cell division, with the activation of cyclin-dependent protein kinases (CDKs) and the degradation of replication licensing factors (RLFs) to prevent reassembly of a pre-replication complex (pre-RC).

Eukaryotic DNA Polymerases

• At least 19 different polymerases are present in eukaryotes, with 5 being studied more extensively.
• The five best-studied polymerases are called a, b, g, d, and e, with different roles in DNA replication and repair.
• Polymerase d is the principal DNA polymerase in eukaryotes, interacting with a protein called PCNA (proliferating cell nuclear antigen).

The Eukaryotic Replication Fork

• The general features of DNA replication in eukaryotes are similar to those in prokaryotes.
• The replication fork involves the formation of Okazaki fragments, initiated by Pol a, and the attachment of PCNA to Pol d.
• The RNA primer is eventually degraded by separate enzymes, FEN-1 and RNase H1, and DNA ligase seals the nicks that separate the fragments.