Genetics Exam 2 Topic 4

How is DNA replication a semiconservative process?

Each new DNA strand has one parental strand and one newly synthesized complementary strand

What are the building blocks of replicat DNA

DNA template, Triphosphate nucleotides Enzymes, a 3’-OH to build on, Energy

What enzymes are involved in DNA replication

DNA Polymerase, Primase, and initiator protein enzymes

What is a characteristic of the origin of replication in bacteria

It is highly conserved throughout time (aka largely unchanged)

What protein causes adjacent unwinding at the origin?

Initiator protein

The unwinding allows the bonding of what proteins?

Helicase and single strand binding proteins

What does helicase do

break H bonds

What do single strand binding proteins do

keeps DNA strands apart

What does DNA gyrase do?

Reduce positive super coil (loosen dna coiling) to allow for unwinding by making double stranded breaks in the DNA

what protein drives elongation?

DNA Polymerases

What are characteristics of DNA polymerases?

They catalyze phosphodiester bonds between 3’ OH and 5’PO4, they always go in the 5’ to 3’ direction, they work off of a template strand, and they require an existing 3’ end of synthesizing strand

What is DNA primase and what does it do?

It is an RNA polymerase, and it synthesizes short stretches of RNA (“primers”)

How many DNA polymerases have been isolated from E.coli?

5

Which polymerase does the most replication in E.Coli?

DNA polymerase 3

Describe the process of DNA polymerase elongating a strand with an RNA primer

DNA polymerase 3 extends DNA strand, DNA Polymerase 1 removes RNA primers and replace with DNA, Ligase Seals the strand

What is the error rate of polymerizing mode and what direction is it?

1 per 100 kb, and 5’-3’

What is the error rate of editing mode, what part of the polymerase is responsible for this process, and what direction is it?

1 per 10 mb, exonuclease, and 3’-5’

Between Polymerase 1 and 3 which has the higher polymerizing rate? Processing of nucleotides until it falls off?

Polymerase 1!

What can DNA polymerase one do that polymerase 3 cant

it can use exonuclease to edit in both directions

What direction is DNA synthesis?

5’-3’

Describe the continuity of DNA synthesis on each strand

Continuous on one strand and discontinuous on the other

Describe the leading strand

Same direction as the movement of the replication fork, single primer, continuous

Describe the lagging strand

opposite direction of the replication fork, synthesized discontinuouslt in a series of okazaki fragments

Describe the things that could occur that to cause termination

Another origin is met on the leading strand, another okazaki fragment is met on the lagging strand, the end of the chromosome is met (eukaryotes), or when the strand is no longer single stranded

What causes the mechanical stopping of elongation

a termination sequence/ or protein blocks the polymerase and the polymerase falls off

Do eukaryotes have one or multiple origins per chromosome

multiple

What does each origin cause the replication of?

One replicon

What does a replicon mean

stretches of DNA that are replicated independently of one another

Why are histone chaperone proteins important?

They remove (because there , transport, and reasseble histones

What protein liscenses origins?

Replication licensing factors

What is the direction of replication in eukaryotes?

Bidirectional

What does protein licensing make sure of?

A way of firing once (the license is LOST after replication begins), each origin only replicates once per cell cycle

Why is it important to remove the histones?

because the DNA is tightly wound up, making it difficult to access the DNA for replication

How many DNA polymerases do eukaryotes have?

at least 15

Do all animals have the same proteins doing the same things?

No they are species specific

How is the end replication problem solved in eukaryotes?

Solved by telomeres and telomerase! Telomeres are broken down, and telomerase adds repeats to elongate the telomeric region, and it makes enough space for another protein to come and elongate the telomerase region to meet the original sythesized DNA

How many repeats do telomeres usually range from?

250-1500 times

What arises when the wrong nucleotide is added

a single nucleotide polymorphism

How many SNPs are produced in each round of synthesis of the human genome

300

What is replication slippage

When a “slip” takes place and a loop is formed on either the synthesizing or the template strand

What can replication slippage in the new strand cause

it causes more repeats in the new strand than the parental strand

What can replication slippage in the parental strand cause

less repeats in the new strand

What does polymerase chain reaction do?

Replicate a segment of DNA in a tube!

What does dideoxy/sanger sequencing do?

replication in a tube of a single gene

What does illumina sequencing do?

Replication on a slide to sequence a genome

Illumina sequencing is also known as…

Next generation or High throughput sequencing

Steps of PCR

Denaturation, Annealing, and elongation

How do the primers affect PCR?

They determine the portion of the DNA that is amplified

What role do ddNTPs play in sanger sequencing?

They make it so that another 3’ OH cannot be added after (growing strand stops synthesizing)

What are the products of sanger sequencing

Differently sized DNA fragments because of the ddNTPs causing the strand to stop

How to visualize sanger sequencing?

On a gel!

How to read a sanger sequencing gel

read it backwards to determine sequence

What does next gen/ high throughput sequencing do?

Sequences all nucleic acids in a sample at the same time by complementary strand synthesis

How does illumnina sequencing work

DNA fragments are tethered to a slide, universal primer is added, dNTPs with flourescence are added on top of each slide and a photograph is taken to see which dNTPs are incorporated(repeat!).

which is the most expensive tpye of sequencing

next gen sequencing

What is the best way to compare a genome across individuals

next gen sequencing

Which is the most time consuming sequencing option

next gen sequencing