DNA Replication: Steps and Enzymes

• DNA replication is the process of copying DNA.
• The learning outcomes for a session on DNA replication include describing the process, the origins of replication, replication forks, key steps, and enzymes involved.

Initiation

• Proteins bind to DNA and open up the double helix.
• DNA is prepared for complementary base pairing.
• Primase builds an RNA primer, which is 5-10 ribonucleotides long.
• DNA polymerase attaches onto the 3' end of the RNA primer

Elongation

• Proteins connect the correct sequences of nucleotides into a continuous new DNA strand.
• DNA polymerase uses each strand as a template in the 3' to 5' direction to build a complementary strand in the 5' to 3' direction.
• This results in a leading and a lagging strand.
• Along one template strand, DNA polymerase III can synthesize a complementary strand continuously by elongating the new DNA in the mandatory 5' to 3' direction.
• The DNA strand made this way refers to the leading strand.
• The other parental strand (5' to 3' into the fork), which refers to the lagging strand, is copied away from the fork.

Termination

• Proteins release the replication complex so synthesis concludes.

Enzymes and other proteins in DNA Replication

• Topoisomerase unwinds and opens the DNA helix.
• Helicase breaks hydrogen bonds between nucleotide pairs to separate DNA strands.
• Single-stranded binding protein (SSBP) keeps separated strands apart.
• Primase synthesizes RNA primers and helps DNA polymerase to figure out where they should begin.
• DNA polymerase III (The Builder) is the main enzyme for DNA synthesis.
• DNA polymerase I removes RNA primers and replaces them with DNA.
• DNA ligase joins Okazaki fragments and connects DNA segments.
• DNA polymerase catalyzes the covalent bond between the phosphate of one nucleotide and the deoxyribose (sugar) of the next nucleotide and starts at the same time for both strands.
• RNase degrades the RNA primer.

Leading Strand Synthesis

• Topoisomerase unwinds DNA, afterwards Helicase breaks hydrogen bonds.
• DNA primase creates a single RNA primer.
• DNA polymerase slides along the leading strand in the 3' to 5' direction synthesizing the matching strand in the 5' to 3' direction.
• The RNA primer is degraded by RNase and replaced with DNA nucleotides by DNA polymerase.
• DNA ligase connects the fragment at the beginning of the new strand to the end of the new strand

Lagging Strand Synthesis

• Topoisomerase also unwinds DNA then Helicase breaks hydrogen bonds.
• DNA primase creates RNA primers, but in spaced intervals.
• DNA polymerase slides along the leading strand in the 3' to 5' direction synthesizing the matching Okazaki fragments in the 5' to 3' direction
• The RNA primers are degraded and replaced with DNA nucleotides
• DNA ligase connects the Okazaki fragments to one another
• Unlike the leading strand, which elongates continuously, the lagging strand is synthesized as a series of short segments referred to as Okazaki fragments.
• For synthesis of the lagging strand, each Okazaki fragment needs priming.
• DNA polymerase I replaces the RNA nucleotides of the primers with DNA, adding them one by one onto the 3' end of the adjacent Okazaki fragment.
• The primers are converted to DNA before DNA ligase joins the fragments
• Along one template strand, DNA polymerase III can synthesize a complementary strand continuously by elongating the new DNA in the mandatory 5' to 3' direction.

DNA Polymerase: Properties, and other Enzymes

• Enzyme that catalyze the covalent bond between the phosphate of one nucleotide and the deoxyribose (sugar) of the next nucleotide
• Cannot initiate synthesis of a polynucleotide but can add nucleotides to the 3' end of existing strand paired with a template strand.
• The initial nucleotide chain acts as a primer
• In cells, the primer is a short stretch of RNA with available 3' end
• Primase, which refers to an an RNA polymerase, links ribonucleotides complementary to template DNA into the primer.
• RNA polymerases can start an RNA chain from a single template strand
• After the primer is formed, DNA pol III adds a deoxyribonucleotide to the 3' end of the RNA primer and continues adding DNA nucleotides to the growing DNA strand based on base-pairing rules.
• The rate of elongation is ~500 nucleotides per second in bacteria, and ~50 per second in human cells.
• Two distinct polymerases active in E. coli: DNA polymerase III, and DNA polymerase I
• Eukaryotes feature eleven different DNA polymerases.
• The strands in a double helix are antiparallel
• Each strand has a 3' end with a free hydroxyl group, and a 5' end with a free phosphate group, attached to deoxyribose.
• Polymerases add nucleotides to the free 3' end of a growing DNA strand.
• A new DNA strand can only elongate in the 5' to 3' direction.
• Helicase untwists the double helix and separates the template DNA strands at the replication fork.
• Topoisomerase helps relieve strain from this untwisting.
• Single-strand binding proteins keep the unpaired template strands apart during replication.
• Helicase unwinds the parental double helix.
• DNA primase synthesizes the primer starting the process.
• Lagging strands undergo transcription in segments in 5' to 3' direction (Okazaki fragments).
• Ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1.
• DNA polymerases cannot initiate synthesis of a polynucleotide
• Removes the primer from the 5 end of the fragment, replacing it with DNA nucleotides
• Synthesis proceeds continuously in the 5' to 3 direction.
• Ligase bonds the 3' end of one fragment to the 5' end of the first fragment.
• Lagging strand is transcribed in segments in 5' to 3' direction (Okazaki fragments).
• DNA ligase connects two Okazaki fragments.
• An origin of replication is where the leading and lagging strands originate from.