Cell Biology Unit 1- Lecture 2

DNA polymerase

enzyme that catalyzes the covelant linkage between a nucleoside triphosphate and a growing DNA strand

adds in 5’ to 3’ direction

proofreading

after nucleotide is bound by DNA polymerase and before it is covalently cross-linked, polymerase checks for good fit

can also check after covalently binding

has nuclease activity to delete incorrect nucleotide

DNA primase

matches RNA nucleotides with DNA on the template strand

gives DNA polymerase a place to start

error prone because it doesn’t work from existing base pairing.

leading strand

gets primed once at the 3’ end and the polymerase synthesizes DNA from 5’ to 3’

lagging strand

gets primed at regular intervals and polymerase stops when it hits the previously synthesized DNA

okazaki fragments

fragments of DNA created by DNA polymerase

nucleases

identify and remove RNA primer

special DNA polymerase then replaces primed area with DNA nucleotides accurately

DNA ligase

repairs remaining tiny defect by adding phosphodiester bond

sliding clamp

protein complex that works with DNA polymerase to keep it attached until it runs into double-stranded DNA

loader attaches it to DNA polymerase

also helps with proofreading

strand-directed mismatch repair

happens when specialized proteins sense a defect, bind to a nearby sliding clamp, and remove the synthesized strand

DNA helicase

uses ATP to move along the DNA helix, physically forcing the helix to unwind and strands to separate

single-strand DNA binding proteins

holds the strands straight and prevent re-winding

DNA topoisomerase

relieves tension and supercoiling by forming temporary strand breaks to create flexibility

strand-directed mismatch repair

addresses any remaining mismatches

telomeres

ends of chromosomes

non-coding region that protects the coding DNA

telomerase

carries an RNA template used to extend the telomere of the parents strand duering replication

leaves room for DNA primase and DNA polymerase to synthesize the lagging strand

allows telomeres to stay about the same length

short temomeres

from aging because telomerase activity gradually decreases over time

can indicate to a cell that it should stop dividing

continuing to divide may cause defects to gene-expressing regions

more divisions means more likely mistakes are made

cancer and stem cells

have more telomerase activity and intentionally keep telomeres longer

DNA damage

will propagate and expand in a cell population

can result from:

• mistakes during replication

• chemicals/radiation causes chemical changes to nucleotides, single-strand breaks or ds breaks

• intentional strand breaks made by cell

strand-directed mismatch repair

done by DNA polymerase

eliminates almost all errors from replication

deamination

loss of amino group, can happen to any nucleotide

ex. deamination changes C to U (not usually in DNA)

U is similar to T, so when DNA polymerase reads it, it will add an A instead of a G

depurination

loss of purine group, so can happen to A or G

ex. depurination removes A, leads to deletion

base excision repair

enzymes remove the offending base only

nucleotide excision repair

enzymes remove surrounding sequence including backbone

double strand breaks

can be formed in response to chemicals and radiation or normal cellular processes

non homologous end joining

the simplest and most common repair mechanism

DNA ligase fuses the ends of DNA around the double strand break- usually leads to local deletion

generally fine because much of genome is non-coding

homologous recombination

more accurate method of repair

uses similar or identical sequence as a template to fix damage

can often use paired chromosome as template for repair

once break is identified, nuclease digest 5’ ends to expose single strands for base pairing

one strand invades the template helix and matches up by base pairing

DNA polymerase is primed by matches basepairs and can start repairing the invading strand

after sufficient repair, invading strand is released and can serve as a template to repair the other damaged strand

cross-over

during this, double strand breaks are intentionally created

process starts as normal homologous recombination, but invading DNA strand isn’t released

DNA synthesis occurs on both damaged strands using both template strands forming structures called holiday junctions, which are eventually cut to leave invading strand with template strand

CRISPR

a Cas enzyme can work together with a small RNA sequence called a guide RNA to create targeted double-strand breaks, which will then be repaired

targeted deletion

can “knock out” a gene of interest by deleting it from genome using CRISPR

targeted editing

using CRISPR to edit a gene of interest or insert something

provide cells with donor DNA as template for homologous recombination repair to insert into genome