DNA Replication

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DNA Replication

• arranged by M. Keffer

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Replication of DNA

• It is crucial that the DNA be able to create
  exact replicas of itself.
• otherwise, every generation of cells would be
  different
• and a child would never look like its parents
• indeed, there wouldnt even be any reason for it
  to look like a human!

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Complementarity

• this is the idea that an A always matches with a
  T, and C and G always match
• this provides the ability for DNA to make an
  exact copy of itself

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the simple

• at its most simple, the double stranded DNA
  separates
• complementary DNA nucleotides (free-floating in
  the nucleus) drift into place
• another enzyme stitches the new NTs into a new
  strand of DNA
• this new strand is COMPLEMENTARY to the original
  (NOT identical to it)

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semiconservative replication

• each daughter strand is HALF OLD, HALF NEW.
• this is likely to be more accurate over the long
  run, when viewed over thousands of generations
• if there is an error here on the new strand
• there is still a correct old version to back it
  up

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conservative replication

• if replication were conservative,
• one daughter strand would be completely NEW
• while the other was completely OLD
• this is NOT THE WAY IT OCCURS.
• this would mean that the parent strands would
  have to separate, act as template, dissociate
  from the new strands, then reassociate with each
  other.
• the semiconservative way is simpler.

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Relax and Unwind...

• DNA is extensively twisted
• gyrase
• (aka topoisomerase)
• picks a starting point and untwists the coil
• then helicase cuts the H-bonds between the
  strands
• this opens a bubble

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Relax and Unwind...

• DNA is extensively twisted
• gyrase (aka topoisomerase) picks a starting point
  and untwists the coil
• then helicase cuts the H-bonds between the
  strands
• this opens a bubble

Gyrase relaxes supercoiling
Helicase breaks H-bonds
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ori C and many bubbles

• there are many places where replication bubbles
  start
• they are triggered by specific sequences on the
  DNA
• sequences are called oriC

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now for the tricky stuff...
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rate of replication

• prokaryotes can add about 500 NTs PER SECOND with
  only one error per billion
• E.coli has about 15 copies of DNAP per cell
• eukaryotes can add about 50 NTs per second
• but eukaryotic cells might have upwards of 50 000
  copies of DNAP per cell
• also there are many many bubbles in eukaryotes
  (therefore many sites of origin)
• replication in euks is occurring simultaneously
  at many sites

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DNAP is a DIMER

• the DNAP actually folds over the lagging strand
• DNAP is actually a dimer (made of two parts)
• the loop allows the DNAP dimer to move in the
  forward direction
• it can replicate both strands at once

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summary all the steps

• 1. starting at oriC, gyrase unwinds the DNA
• 2. helicase breaks H-bonds between strands
• 3. bubble develops
• 4. primase adds RNA primer(s)
• for leading strand (on the left), primers added
  at the bottom fork
• for lagging strand (on the right), primers added
  NEAR the top fork
• 5. DNA Polymerase attaches to primed section,
  begins replication 5 ---gt 3
• leading strand moves UP the LEFT in CONTINUOUS
  way
• lagging strand moves DOWN the RIGHT --
  DISCONTINUOUS (makes Okazaki fragments))
• 6. DNA ligase stitches nucleotides together and
  removes RNA primers

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Replication errors

• errors are made, but there are mechanisms to fix
  them
• estimated that only 1 in 1010 errors remain after
  repair mechanisms have occurred
• if there was ONE typo error in your whole
  textbook (which is about 4 x 106 characters),
  that would be about 2000 times more errors than
  in a mammalian cell!
• after that there is still a large possibility
  that that one error might be in a non-coding
  region of DNA
• (most of DNA is non-coding, actually)
• or, the error might not make a difference when it
  comes to translation (can you guess why?)
• (because the genetic code is degenerate -- there
  is more than one code for each amino acid (in
  most cases))

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Polymerase and Ligase
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• at the bottom fork, DNA polymerase starts
  adding NTs
• it goes UP the LEFT strand
• this is going in the SAME DIRECTION unwinding
• DNA ligase stitches the new NTs together

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DNAP
lig
5
3
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the 5 ---gt 3 problem

• by some quirk of nature, new NTs can ONLY be
  added at the 3 end of a strand.
• that means that a strand of NTs can ONLY grow in
  the 5 ---gt 3 direction
• NOT the other way!
• this presents a slight problem when we look at
  the other strand

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the 5 ---gt 3 problem

• by some quirk of nature, new NTs can ONLY be
  added at the 3 end of a strand.
• that means that a strand of NTs can ONLY grow in
  the 5 ---gt 3 direction
• NOT the other way!
• this presents a slight problem when we look at
  the other strand

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the 5 ---gt 3 problem

• by some quirk of nature, new NTs can ONLY be
  added at the 3 end of a strand.
• that means that a strand of NTs can ONLY grow in
  the 5 ---gt 3 direction
• NOT the other way!
• this presents a slight problem when we look at
  the other strand

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the 5 ---gt 3 problem

• by some quirk of nature, new NTs can ONLY be
  added at the 3 end of a strand.
• that means that a strand of NTs can ONLY grow in
  the 5 ---gt 3 direction
• NOT the other way!
• this presents a slight problem when we look at
  the other strand

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ADD ONLY TO 3 END!!

• new NTs can ONLY be added at the 3 end
• new NTs can ONLY be added at the 3 end!!!
• therefore DNAP goes DOWN the RIGHT strand and UP
  the LEFT strand.

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the lagging problem...

• with the leading strand (on the left), there is
  only one priming site
• however, with the lagging strand (on the right),
  there must be MULTIPLE priming sites
• note that unwinding happens in ONE direction
  (up)
• on the lagging strand, replication is
  DISCONTINUOUS (must start and stop)

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fragment stitching

• DNA ligase stitches the pieces together
• these fragments are called
• RNA primers are also removed at this point by
  ligase)
• fragments are about 150 NTs long in mammal cells

OKAZAKI FRAGMENTS
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summary the medium level of difficulty
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5

• DNA replication uses DNAP and ligase
• replication must add new nucleotides to the 3
  end of the chain
• the new strand forming on the right side is
  called the leading strand and is continuous
• the new strand forming on the left is called
  the lagging strand and is discontinous
• the fragments formed on the left are called
  Okazaki fragments
• the O.F. are stitched together by DNA ligase.

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3
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RNA primers

• another quirk
• replication can ONLY occur when started with a
  DOUBLE-STRANDED SEGMENT
• this is probably to protect against replication
  of foreign DNA
• therefore PRIMERS must be added
• primers are RNA, and are made by PRIMASE