Lecture 20: DNA replication and repair

Replication converts what to what

DNA -> DNA

DNA replication two models

semiconservative and conservative

Semiconservative model

hybrid of duplex old and new strand

Conservative model

duplex of only old or only newly synthesized DNA

DNA synthesis is performed by

DNA polymerase

DNA synthesis requires

template strand to copy, primer strand with 3’ OH, dNTP substrates

DNA synthesis is catalyzed by

nucleophilic attack by 3’ OH, phosphodiester bond formation, 5’ to 3’ synthesis

DNA synthesis always occurs from what to what

5’ to 3’

_____ drive DNA synthesis

triphosphates

Triphosphate cleavage is what makes what reaction work

polymerization

In DNA synthesis, ____ ____ in active site balance negative charge

magnesium ions

Primer strand

5’-> 3’

Template strand

3’ -> 5’

In DNA synthesis, basepairing directs choice of ____

dNTP

What do magnesium ions do in DNA synthesis

balance negative charge in active site

____ residues hold the Mg2+ ions in place

asparagine

DNA polymerase function

reads the base on the template strand and inserts the corresponding pair

___ ___ is VERY well designed to ensure base pairs are actually corresponding

active site

Base pair ____ (has to do with shape) is important

geometry

DNA polymerases insert one incorrect nucleotide for every ___ to ____ correct nucleotides

10^4 to 10^5

exonuclease activity function

proofreading

Exonuclease function

remove nucleotide via phosphodiester bond hydrolysis

Exonuclease activity is from

3’-> 5’

Exonuclease steps

Polymerase shifts mispaired bases to exonuclease site for removal After removal, it is shifted back and you have effectively deleted the last one

All DNA Pols have ___ -> ____ _____ to repair

3’-> 5’ exonuclease

Pol I is the only polymerase with ___ -> ____ _____ activity

5’-> 3’ exonuclease

DNA replication three major steps

initiation, elongation, termination

What step is the commitment step

initiation

DNA Replication initiates at

replication origins

DNA Unwinding Element (DUE) has a high amount of ___ because they have fewer ____ ______so it is easier to separate them

AT, hydrogen bonds

DNA Binding Sequences where DnaA protein binds to the _____ sequence (origin of replication, where the duplex splits)

oriC

What binds to specific site in origin and recognize oriC sequence

DnaA

DnaB protein

helicase, unwinds DNA

Elongation/polymerization uses

DNA polymerase III

DNA polymerase synthesis only in this direction

5’ -> 3’

Leading strand synthesis is

continuous

Leading strand synthesis is in the direction of

fork movement

Lagging strand synthesis is

discontinuous

lagging strand synthesis is in the direction of

opposite of fork movement

Replisome

Collection of proteins used for replication

Primase used on _______ strand to initiate each okazaki fragment

lagging

Primase lays down

RNA primers

Lagging strand synthesis direction relative to replication fork and fragments

3’ -> 5’, has okazaki fragments

For leading strand, primase synthesizes an RNA primer where

at the origin

For the lagging strand, primase synthesise an RNA primer

for each Okazaki fragment

dNTPs are added by

DNA polymerase III

DnaB/helicase

unwinds

SSB

stabilize strands

DNA gyrase

relieves strain by unwinding supercoiling

RNA primers are removed by ____ ____ _____

DNA polymerase I

Nicks in Okazaki fragments are closed by

DNA ligase

Made of two subunits that encircling the DNA

sliding clamp

Sliding clamp is what causes DNA Pol III to be highly

processive

Clamp loader helps enzymes initially attach what to DNA

sliding clamp to the DNA (uses ATP!)

What makes up the beta sliding clamp

two beta subunits

What removes RNA primers

DNA Pol I

DNA Pol I has special __ -> ___ exonuclease activity that allows it to be awesome at removing the RNA primers

5’ -> 3’

DNA pol III has a

clamp loader

DNA polymerase I has what direction exonuclease activity and polymerase activity

both 5’-> 3’

Elongation

removal of RNA primers by Pol I

Elongation- RNA primers replaced with

DNA

5’ -> 3’ exonuclease activity function

removes RNA

5’ -> 3’ polymerase activity

fills in with DNA

DNA ligase

sealing the nick

Phosphodiester bond formation

1) adenylation of enzyme 2) activation of 5’ phosphate 3) nucleophilic attack by 3’ OH

Termination of replication is marked by

ter region

Replication fork stops at what region

terminus

Decatenates (unlinks) the two chromosomes

Toposiomerase IV

Replication in eukaryotes replication rate is _____, and the chromosomes are ____

slower, longer

Eukaryotic chromosomes are

long and linear

____ ____ __ ____ are necessary to replicate large chromosomes

multiple origins of replication

Chromosomes must be replicated ___ ___ per cell cycle

only once

What assembles at the eukaryotic origin

pre-replicative complex

Chromosomes are made of

two telomeres and a centromere

Telomerase adds on to _____ ends

chromosomal

Telomerase synthesis DNA from a ___ template

RNA (reverse transcriptase)

The template is an ____ molecule that is part of the enzyme

RNA

Telomerase is a ________ (______)

ribonucleoprotein (RNP)

Reverse transcription

RNA-dependent DNA synthesis

Reverse Transcription Application in HIV- ____ is able to insert its genome directly into eukaryote genome

retrovirus

retrovirus is able to insert into ___ ___

eukaryotic genome

Once retrovirus is able to inert into the human genome, it is ___ ___ to remove

very hard

Current solution to HIV

keep viral reproduction from going into the body

______ are proteins in the genome that have a reverse transcriptase function and can add to the human genome

retrotransposons

The human LINE-1 Retrotransposons contribute to

age associated diseases

Part of LINE-1 RNP, has a piece of ______ and inserts copies of itself into the ______

DNA, genome

LINE-1 RNP- Also ______ ______ _____in the immune system

deactivates inflammatory responses

Mutation

a permanent change in the DNA sequence

Mutations can be three things

silent, deleterious, advantageous

Silent mutation- how does it influence gene function

no effect on gene function

deleterious mutation- how does it influence gene function

impairs gene function

advantageous mutation- how does it influence gene function

enhances gene function

Mutations can lead to

genetic diversity, cancer in somatic cells, birth defects in germ cells

Mutations can be caused by

mistakes in replication, DNA damage

Base substitution examples

transition, transversion, base insertion, base deletion

Transition mutation

pu -> pu or py -> py

Transversion mutation

pu <-> py

Types of DNA damage

deamination, depurination, UV irradiation (thymine dimers), alkylation

Deamination converts cytosine to

uracil

Deamination converts 5-methylcytosine to

thymine