L11: DNA Replication in Vivo - Inside Cells Flashcards
What is the function of DNA replication?
- DNA Encodes all of the proteins and RNA (rRNA & tRNA) in the cell to allow for proper cell function
- New cells will need a copy of the genome (all DNA)
Explain the concept of semiconservative DNA replication?
- Each new DNA duplex consists of one strand that was originally part of the parental duplex and one newly synthesized strand (daughter strand).
What is the enzyme that plays a big role in DNA replication?
- DNA polymerase
What are some key properties of DNA polymerase?
- it “reads” the sequence on a template and links nucleotides together
- ex: reads the template 3’ to 5’ and synthesizes new DNA in the direction 5’ to 3’
- cannot start on its own; it must always start from an existing 3’ OH end of an existing template (a primer; RNA in the cell)
What are the steps of DNA polymerization?
- Incoming nucleotides are accepted if they correctly base-pair w/ the template.
- The 3’ OH of the growing strand attacks the high-energy phosphate bond of the incoming nucleotide, providing energy to drive the reaction.
What provides energy to the polymerization reaction?
- Activated monomers (triphosphates) are used to provide energy to drive the reaction
What are the enzymes involved in DNA replication?
- Helicase
- Primase
- DNA Pol. I
- DNA Ligase
- Topoisomerase
- Single-Stranded Binding Proteins (SSBPs)
- DNA Pol. II
What is the function of Helicase?
- Breaks H-bonds
- Unwinding DNA double helix
What is the function of Primase?
- Synthesizes RNA primers on leading and laggings strands
What is the function of DNA Pol. I?
- Replaces RNA primers with DNA nucleotides
What is the function of DNA ligase?
- Catalyzes phosphodiester bond formation
- Join all Okazaki fragments
- joins leading and lagging strands at the origin of replication
What is the function of Topoisomerase?
- Relaxes supercoiled DNA
- Relieves stress on the winding helix
What is the function of Single-stranded binding proteins (SSBPs)?
- Coats single-stranded DNA
- Keeps the single-stranded DNA apart
What is the function of DNA Pol. II?
- Synthesizes DNA 5’ to 3’ on leading and lagging strands
What’s the difference between replication in PROKARYOTES and EUKARYOTES?
- Prokaryotes: Bacterial cells can replicate in a circle - both directions. The ends will meet up, and it usually has 1 origin of replication (OriR)
- Eukaryotes: DNA can be big and linear so need more than one origin of replication (OriR)
What are the 3 problems DNA must solve in order to replicate DNA?
- Separate the DNA strands only a little at a time
- Can’t separate an entire genome - very messy
- Make primers for DNA Polymerase
- can’t make a DNA primer without a DNA primer
- Cell makes an RNA primer - can start without a 3’ OH
- Allow synthesis to happen simultaneously from the two template strands
- DNA strands are anti-parallel yet DNA polymerization has to occur in the 5’ - 3’ direction
- Synthesis has to happen in opposite directions
- DNA strands are anti-parallel yet DNA polymerization has to occur in the 5’ - 3’ direction
Describe what happens as the replication fork opens.
- At first, all is awesome - both strands are synthesized at the replication fork (although in opposite directions)
What is the problem encountered with the replication fork?
- As it opens further, the “top” strand has DNA polymerase moving (synthesizing) to the right and leaving a gap at the fork.
What is the solution to this problem at the replication fork?
- Add another Okazaki fragment to join the first fragment to fill in the gap
What will the cell have to do as DNA replication proceeds?
- Remove RNA primers and replace with nucleotides that are DNA
- Link all the fragments together to form one continuous strand
Discontinuous vs. continuous synthesis.
- Discontinuous: lagging strands
- Continuous: leading strands
Describe the leading strand.
- A daughter strand that has its 3’ end pointed toward the replication fork
- can be synthesized as one long, continuous polymer
Describe the lagging strand.
- Grows away from the fork
- Daughter strand is synthesized in relatively short, discontinuous pieces
- 5’ end pointed toward the replication fork
- Okazaki fragments: the short pieces in the lagging strand.
