Lecture 14

Textbook Notes

15.3 A Model for DNA Synthesis

• DNA polymerase — polymerizes deoxyribonucleotide monomers into DNA, catalyzes DNA synthesis (5’ → 3’)

• DNA synthesis starts with adding 2 phosphate groups to deoxyribonucleotide monomers to form dNTPs

• origin of replication

  • eukaryotes have multiple oris along each chromosome, replicating in either direction (bidirectional)

• replication fork — Y-shaped region where the parental DNA double helix is separated into single strands and copied

How is the Helix Opened and Stabilized

• in bacteria, a specific set of proteins recognizes the origin of replication on a chromosome, and strands near the ori are separated

• binding of enzyme DNA helicase to one of the single strands near each of the forming replication forks

• in eukaryotes, DNA helicase is loaded onto double-stranded DNA

• DNA helicase uses the energy of ATP hydrolysis to separate the strands

• single-strand DNA-binding proteins (SSBPs) attach to separated strands to prevent them from snapping back into the double helix

• topoisomerase (DNA gyrase) cuts DNA, allows it to unwind, and rejoins it in order to release the pressure of twisting from the DNA helicase

How is the Leading Strand Synthesized?

• DNA polymerase can synthesize DNA only in the 5’ → 3’ direction

• DNA polymerases cannot start synthesis from scratch on template strand

  • DNA polymerase can only extend from the 3’ end of an existing strand than is hydrogen bonded to template

  • RNA primer added by primase, one type of RNA polymerase, which can start synthesis from scratch

• leading strand (continuous strand) — DNA strand synthesized toward opening replication fork

  • its synthesis can proceed continuously in the direction of the moving replication fork

How is the Lagging Strand Synthesized?

• lagging strand (discontinuous strand) — synthesized in a direction away from the moving replication fork

• okazaki fragments — short DNA fragments attached to RNA primers

  • in bacteria, DNA polymerase III dissociates from 3’ end of an okazaki fragment when it reaches RNA primer of the next fragment

  • DNA polymerase I then attaches to 3’ end, removing the RNA primer and replacing ribonucleotides with deoxyribonucleotides

  • DNA ligase catalyzes a phosphodiester bond between 3’ and 5’ ends, closing up the backbone

15.4 Replicating the Ends of Linear Chromosomes

• telomere — region at the end of a eukaryotic chromosome

• single-stranded DNA at the end of lagging strand is degraded, shortening the replicated chromosome

Telomerase

• telomeres are made of short stretches of bases repeated over and over

• telomerase replicates telomeric DNA by catalyzing the synthesis of DNA using an RNA template

  1. lagging strand leaves unreplicated single-stranded “overhang” at 3’ end

  2. telomerase binds to 3’ end of template strand, catalyzing the extension of overhang to end of template region of its RNA molecule

  3. then shifts down, adds another copy, over and over again

  4. DNA polymerase can now do its job

• telomerase doesn’t work for a lot of cells, only in gamete cells or stem cells

15.5 Repairing Mistakes/DNA Damage

• DNA polymerase inserts an incorrect base 1 in 100,000 bases

• exonucleus active site — where incorrect base gets removed

• mismatch repair — error correction

• nucleotide excision repair — removes damaged region in one strand of DNA and replaces it

Lecture Slides

• eukaryotes must initiate replication in multiple locations to finish prior to cell division

• strands are separated by helicase enzymes, and are kept single-stranded by single-stranded DNA binding protein

  • DNA helicase unwinds the strands

  • SSB proteins holds the strands apart

  • topoisomerase (not a part of replication bubble) — cuts DNA, lets it unwind, puts it back together

• DNA strand synthesis

  1. incoming dNTP is hybridized to parental template

  2. phosphodiester bond formed with 3’ end of chain

  • first, polymerase checks whether the sugar (ribose vs deoxyribose) is correct, then checks whether the hydrogen bonds are matching, and then breaks off the phosphate groups, releasing energy that creates a phosphodiester linkage to the rest of the phoshate-sugar backbone

  • synthesis reaction that adds a nucleotide to 3’ end is catalyzed by polymerases, and is an endergonic reaction

  • the phosphates hydrolyzed off feed synthesis reaction

• DNA replication is bidirectional

  • new DNA needs to be synthesized on both strands on both sides of the ori (5’ → 3’)

  • but synthesis only occurs in the 5’ to 3’ direction, and the new strands have to be antiparallel to the template

• to solve this problem, DNA synthesis on one side of the ori begins at the ori and proceeds normally

  • called the leading strand, or continuous strand

  • continues uninterrupted, moving with replication fork

• but DNA syntehsis on the other side of the ori starts a short distance away from the ori and works back toward the ori

  • called the lagging strand

  • small fragments of DNA are called Okazaki fragments

• this way, all synthesis occurs 5’ → 3’

• eukaryotes don’t use topoisomerase the same way that bacteria do, because we have linear chromosomes

• leading/lagging strand DNA synthesis is called semi-discontinuous replication

• problem #2: DNA polymerases cannot start a new DNA strand from scratch

  • they absolutely require a free 3’-OH group to which to add the incoming dNTPS — they need primers

• solution: RNA synthesizing enzymes can use a single-stranded DNA template to make an RNA strand from scratch

  • the special DNA-dependent, RNA-synthesizing enzyme used in DNA replication is called primase

  • primase creates a short (5-15 nucleotide) strand of RNA opposite a single-stranded DNA template called a primer

  • this gives the major DNA-dependent, DNA-synthesizing enzyme (DNA polymerase III in E. coli) what it needs—a free 3’-OH group

  • “priming the pump” of DNA synthesis

• every fragment is primed

• RNA primers must now be removed, or the genome would be littered with RNA bases

• RNA nucleotides removed and replaced with DNA nucleotides by DNA polymerase !

• backbone of new chain has “nicks” in it where no covalent linkage exists between nucleotides

• these nicks are sealed by DNA ligase, creating intact double-stranded DNA

• important terminology:

  • “X”-dependent “Y”-synthesizing enzyme

    • “X” = what it uses as a template

    • “Y” = what it is making

  • an enzyme that degrades (hydrolyzes) a phosphodiester linkage = a nuclease

  • a nuclease that hydrolyzes nucleic acid from the end of a chain = an exonuclease

  • a nuclease that hydrolyzes nucleic acid internally (i.e., not at one end or the other) = an endonuclease

  • if an exonuclease starts at the 5’ ed, working toward the 3’ end, it is called a 5’-3’ exonuclease

  • if an exonuclease starts at 3’ end, working toward 5’ end, it is called a 3’-5’ exonuclease

  • DNA polymerase I’s ability to remove primers is due to its 5’-3’ exonuclease activity, which is a separate enzymatic activity from its DNA synthesizing ability

• proofreading: an example of 3’-5’ exonuclease activity

  1. DNA polymerase adds a mismatched deoxyribonucleotide

  2. mismatch is displaced into an exonuclease site and removed

     • “backing up” 3’-5’

  3. polymerase addes the correct deoxyrribonucleotide