DNA polymerases in DNA repair Flashcards
DNA replication proteins
DNA replication machine
DNA replication definition
Process of copying DNA before cell divides
DNA replication key words (when explaining the process of DNA replication, these points must be met, start with definition above) [add more detail in an exam answer but must include these key points]
Strands separate, (each strand must act as a template) act as 2x template strands, **complementary base pairing (only copies if it has something to copy off) each template is used by proteins/DNA replication machine to carry out the process
-(where it happens) Ori (origin of replication site, the origin of where this replication occurs) site (1x prokaryotic, 10,000 eukaryotic) but when the process of DNA replication occurs it starts at all 10,000 sites in eu cells at the same time, the proteins that star this process start all at once, only 1 in pro cells, so only one starting site etc…if you mention the ori site, specify that there is 1 in pro and contrasts to 10,000 in eu
-Y-shaped replication in fork (active DNA synthesis) (DNA does not completely separate, a little break happens to allow the replication to occur, active synthesis, active replication
-Bi-directional (the replication bubbles grow out tp the left and to the right, 2 separate events going on on each replication fork a left and a right, /synthesis in 5’-3’ direction (because of phospherdiester bonds it is always in a 5’-3’ direction, 2 phosphates are removed from free nucleotide base, in polymerisation process, when referring to poly we refer to polymers being made by the monomer units in the phospherdiester bonds)
-leading strand - continuous (one strand will be 5’-3’ and the other will be anti-parallel ie 3’-5’, leading strand is 5’-3’ and lagging strand is 3’-5’)
-lagging strand - discontinuous
Proteins critical for DNA replication
- DNA polymerase (most import ones, several of them, they polymerise, can have other functions in DNA repair and checking to ensure that the correct nucleotide as been added to the nucleic acid)
- Helicase (breaks down hydrogen bonds (base pairs) at Ori sites, A-T rich sites, t ori sites there is more A-T bonds than G-C bonds as they only have 2 bonds whereas G-C has 3 bonds so A-T is easier to break open so there’s more of them)(think of zipper on a jacket)
3.ligase (acts like a glue, stitches/repairs phospherdiester back bone if its not completely sealed together)
ending with -ase
protein with catalytic function
Leading strand
continuous so ligase is not needed
Lagging strand
discontinuous needs ligase as it needs to be sealed at all time
Polymerisation active (-ase due to catalytic activity)
catalyses the addition of nucleotides to the free 3’ OH on growing DNA strand. Nucleotides enter reaction as nucleoside triphosphate. Breakage of phosphoanhydride bond (indicated by *) in incoming nucleoside triphosphate releases large amount of free energy and thus provides energy for reactionD
DNA polymerisation (Pol) is so accurate that it
makes only ~1 error in every 10 million (10^7) nucleotide pairs it copies
This is because DNA polymerisation can correct its mistakes (self-correcting), it has error-correcting activity- proofreading
can remove it and replace it with polymerisation activity with the correct one
read from
5’ to 3’
Removed in reaction
pyrophosphate
DNA polymerases
add incoming deoxyribonucleic triphosphate
DNA polymerases (2 in bacterial DNA replication)
Before DNA pol adds nucleotide to growing dna chain, it checks whether the previous nucleotide added is correctly base-paired to the templates strand. there are 2 outcomes
Yes and No
Yes
correct base-pairng: DNA pol proceeds and adds next nucleotide
No
Incorrect base-pairing: DNA pol removes misfired nucleotide by cutting phospherdiester bond, releases nucleotide and dies again to add correct nucleotide
DNA polymerase III
is the main replicating enzyme in bacteria and possess two enzymatic activities
A 5’-3’ (forward, [as its only ever one way]) DNA polymerase activity, requiring a 3’ primer site and a template strand
A 3’-5’ (reverse) exonuclease activity that mediates proofreading (goes back to forward activity if incorrect pair is present, and tries again, knows its incorrect when the base can’t hydrogen bond, will be a distortion in the geometry of DNA an this is detected and knows it has added the correct base, removes incorrect nucleotide selects and new nucleotide and starts again)
DNA polymerase I (can do what DNA polymerase III can do but can also do reverse ad forward exonuclease activities)
replaces RNA in primer with DNA and possesses three enzymatic activities:
A 5’-3’ (forward) DNA polymerase activity, requiring a 3’ primer (nucleotide with an OH group there to start activity) site and a template strand
A 3’-5’ (reverse) exonuclease activity that mediates proofreading, (goes back to forward activity if incorrect pair is present, and tries again, knows its incorrect when the base can’t hydrogen bond, will be a distortion in the geometry of DNA an this is detected and knows it has added the correct base, removes incorrect nucleotide selects and new nucleotide and starts again) (same as DNA polymerase III)
A 5’-3’ (forward) exonuclease activity mediating nick translation (during DNA repair
Structure and function of DNA polymerases
search for diagram
looks like right hand, in shape
5’-3’ polymerisation domain (forward)
3’-5’ exonuclease domain (reverse)
fingers
thumb
palm
_____ fold over on each other to form domains
where thumbs and fingers are is polymerisation domain
where palm is is the exonuclease domain
template strand gets polymerised
exonuclease domain is only needed if an incorrect base pair has been added (2x purines, or 2x pyrimidines can only have 1 of each) if this happens, the exonuclease domain removes incorrect base, and polymerisation domain goes again to add correct base
DNA polymerase I
Main type in eukaryotic cells
DNA polymerase III
Main type in prokaryotic cells
DNA polymerase relies on short lengths of RNA primers to make new DNA
DNA pol can only join nucleotide to a base-paired nucleotide in a DNA double helix: it cannot start a completely new DNA strand
Different enzyme needed to start new DNA strand
This enzyme des not synthesise DNA, but makes a short length of RNA, using DNA strands template
Short length of RNA (5-10 nucleotides) is base-paired to template strand and provides a base-paired 3’ end as starting point for DNA pol
This short segment of RNA = primer and is synthesised by the enzyme primase
Leading strand: only 1 primer required (at replication origin)
lagging strand:
Role of primers n synthesis of leading & lagging strands
lagging strand contains a primer on each strand
primer is converted to deoxyribonucleocide
Completion of leading and lagging strands
To produce a continuous new DNA strand ad remove RNA nucleotides (primer) from the leading strand and the many separate pieces of RNA (primers) and DNA made on lagging strand, 3 additional enzymes are needed:
Nuclease: breaks apart the RNA primer
DNA polymerase I (in bacteria) replaces RNA with DNA
DNA ligase joins 5’ phosphate end of one new NDA fragment to 3’ OH end of the next (ATP
Helicase
enzyme that uses energy of ATP hydrolysis to speed along DNA and separate strands of parental DNA double helix ahead of the polymerase (breaks the H bonds between base pairs)
