DNA Replication & Repair Terms | Biology Study Guide

What is the difference between a ribonucleotide and deoxyribonucleotide, chemically?

A ribonucleotide has a hydroxyl at position 2 on the ribose. A deoxyribose has a hydrogen at position 2 instead.

What is the result of this?

DNA is much more chemically stable than RNA. The presence of the hydroxyl at position 2 on the ribose contributes to instability in RNA.

Which bases is DNA made of?

dATP, dGTP, dCTP, dTTP

How do you tell the 5' end from the 3' end of a DNA strand?

The phosphate group extends from carbon 5 of the ribose ring, so this is the 5' end. The 3' end is the free hydroxyl of carbon 3 on the ribose ring.

What types of bonds occur in DNA?

phosphodiester bonds join adjacent deoxyriboses

In what direction is DNA made?

5' to 3'

Why are the bases inside and the phosphodiester backbone outside?

Phosphates and sugars are hydrophilic. Bases are hydrophobic.

What takes more energy to break, G-C or A-T? Why would a scientist care?

G-C pairs require more energy to break, because G-C pairs are held together by 3 hydrogen bonds while A-T pairs are held together by 2. This is important in transcription

What do we mean when we say DNA is antiparallel?

The 5' end of one strand is opposite the 5' prime end of the other strand.

What is the most common form of DNA? What other forms are there and what are the circumstances where we might see them?

The B-form is most common: bases are paired in the middle, phosphates and sugars face the outside, and there is a major and minor groove. The A-form is another right-handed helix orientation but is more compact than B-form. A-form is found where DNA must bend. The Z-form is a left-handed helix orientation with no pronounced major and minor groove and a longer distance between bases. It is found in chromosomes. Both B-form and A-form DNA may be used to signal that there is a gene nearby in the DNA strands.

What are the major and minor grooves?

Major and minor grooves are grooves in DNA that arise as a result of the double helix. These grooves are used as binding sites by proteins.

How many base pairs (bps) per turn in B-DNA?

10.5

How does DNA get compacted?

Histones help to coil up the DNA

What do we mean by semiconservative replication of DNA?

Each replication results in two strands of DNA, and each strand contains one newly synthesized and one old strand. Each daughter cell thus gets one strand from the parent and one new strand.

What enzyme has the job of making new DNA (generic name for it)?

DNA polymerase

What are the leading and lagging strands? Why does replication have to work this way?

The leading strand, which runs in the 5' to 3' is replicated by continuous replication. The lagging strand is replicated by discontinuous replication. This occurs because DNA polymerase must synthesize new DNA in the 5' to 3' direction.

What is a primer in DNA replication and what is it made of? What enzyme makes it?

A primer is a short piece of RNA that is then grabbed by DNA polymerase and extended by putting DNA on to make the complementary strand. It is made by primase.

What are Okazaki fragments?

The short, discontinuous segments of primer and DNA that make up the complementary strand being formed against the lagging strand.

What direction does DNA polymerase go? What type of bond does it catalyze?

5' to 3' only. Phosphodiester bonds.

What enzyme reads the DNA strand to know what bases to put in during replication?

DNA polymerase

What is elongation?

What is "proofreading?"

A DNA polymerase runs 5' to 3' must read the base in the opposite strand and select the proper base to add to the new strand. Then, it also has 3' to 5' exonuclease activity to read base pairs just added for correctness and remove them if mismatched. Some 'error prone' DNA polymerases lack the proofreading ability; many are viral. E.g. HIV has a polymerase called reverse transcriptase with an error rate of ~1 in 10,000.

What happens when the polymerase puts in an incorrect base? What is this activity called?

The polymerase 'sees' the error and stops. It then uses 3' to 5' exonuclease activity to bread the phosphodiester bond it just made. This is proofreading.

What proteins make replication happen efficiently? What are their jobs?

Helicase stays ahead of the DNA polymerase, separating the two strands of the helix.

A topoisomerase such as DNA gyrase prevents knot formation by releasing tension by clipping DNA strands and then reattaching them.

RNA primase lays down RNA primers for DNA polymerase to use to perform elongation.

Single strand binding (SSB) proteins protect single strands of DNA and interact with DNA pol3

What is the replication fork? How many of them will occur in bacterial chromosome?

DNA pol3 in bacteria has what activities?

DNA polymerase 3 (pol3) replicates the majority of the genome in E. coli. It is a dimer. It has both a 5' to 3' activity and 3' to 5' proofreading activity.

How are knots in DNA replication dealt with or prevented?

Topoisomerases. Type 1 topoisomerases clip one strand and allow unwinding to occur. Type II topoisomerases clip both strands. DNA gyrase is a Type II topoisomerase.

How quickly do bacteria replicate their DNA?

- E. coli - replicate entire genome in 20min

What types of topoisomerase exist? Which type is gyrase?

Topoisomerases can relieve or create tension in DNA depending on purpose. Gyrase is a Type II topoisomerase.

What is the importance of single-stranded binding proteins?

SSBs protect unwound strands of DNA, which are vulnerable to damage. SSBs also interact with DNA pol3, helping replication to proceed efficiently.

What is the job of the sliding clamp in prokaryotes (or proliferating cell nuclear antigen, PCNA, in eukaryotes)?

The sliding clamp, or beta clamp, hangs onto DNA on one side and DNA pol3 on the other side. Its job is to hold DNA pol3 onto the DNA. This is what allows DNA pol3 to have high processivity

What is the chemical process of the formation of the phosphodiester bond in DNA elongation?

DNA polymerase catalyzes the joining of an incoming nucleotide onto the 3' end of an existing nucleotide. A phosphate on the incoming nucleotide is attacked by the oxygen of the hydroxyl on the ribose sugar of the other nucleotide. The incoming nucleotide loses pyrophosphate and a phosphodiester bond forms.

How is the RNA primer removed?

By the third catalytic activity of DNA pol1.

What does DNA pol1 do?

Removes RNA primers from the lagging strand and fills the resulting gaps between Okazaki fragments with DNA.

DNA pol1 has 'third catalytic activity': it is an exonuclease with 5' to 3' activity. This allows DNA pol1 to remove RNA primers from the lagging strand.

While DNA pol1 is removing RNA ahead of itself, it is acting as a 5' to 3' polymerase to fill the gaps with DNA.It does not stay attached to DNA for long, usually under 100 nucleotides; it is "progressive."

What is supercoiled DNA? Why would it be useful? What enzyme could help it supercoil?

Supercoiled DNA is wound tighter than the 'relaxed circle' of B-form DNA. It has tension, causing it to wrap up on itself. Supercoiled DNA takes up less space. Topoisomerases facilitate supercoiling.

What are writhe and twist? How do we get the linking number?

Creation of superhelixes relieves tension on supercoiled DNA strands. One turn (i.e. 10.5 bps per turn) is a twist. Writhes count the number of times the double helix crosses over itself. The linking number is the sum of twists and writhes.

If supercoiling is present, the linking number will not equal the number of twists.

How does polymerase chain reaction (PCR) work? What are the steps? What do you get as a result?

PCR can be used to amplify trace amounts of DNA and/or make many copies of a segment of DNA of interest.

Step 1: DNA denaturation. DNA polymerase requires separated strands to work, so the sample is heated to near boiling to break the hydrogen bonds.

Step 2: Cool mixture and add synthetic DNA primers (typically about 20 nucleotides long) and dNTPs (dATP, dCTP, dGTP, dTTP). The primers form complexes with their complementary sequences in the sample DNA.

Step 3: Warm mixture to allow replication to occur. The primers determine the replication locations in DNA. As the strands re-separate, DNA polymerase does its work.

Repeat the process for 30-40 cycles.

Because of the high heat involved in the PCR process, a DNA polymerase called Taq polymerase from a thermophilic bacteria is used.

Contrast prokaryotic and eukaryotic DNA replication.

Prokaryotic DNA replication:

Occurs inside the cytoplasm

Only one origin of replication per molecule of DNA

Origin of replication is ~100-200 nucleotides long

Replication occurs at one point in each chromosome

Initiation is carried out by DNAA and DNAB

Topoisomerase is needed

Replication is very rapid

Eukaryotic DNA replication:

Occurs inside the nucleus

Have many origins of replication in each chromosome

Each origin of replication is formed of about 150 nucleotides

Replication occurs at several points simultaneously in each chromosome

Initiation is carried out by the Origin Recognition Complex

Topoisomerase is needed

Replication is comparatively slow

Why do you think that eukaryotes have multiple replication forks?

Eukaryotic chromosomes are linear, not circular

What is the origin ("oriC")?

The origin of replication is a specific sequence of DNA that is recognized by the replication proteins. In E. coli, the sequence is called oriC.

How fast do eukaryotic cells replicate their DNA?

~24 hours

Why do you think that origins are rich in A-T base pairs?

Because A-T base pairs only have 2 hydrogen bonds each instead of 3. Opening the strands in an area rich in A-T base pairs therefore takes less energy.

What is the problem that linear chromosomes have at their ends?

Every time a linear DNA strand is replicated, a little bit is lost when an RNA primer is removed at the end and can't be replaced.

What "one special trick" do cells use to deal with this? What is the enzyme?

Cells have repeating DNA sequences that are noncoding at the ends of strands. Telomerase keeps these ends intact. Telomerase is most active in embryonic cells and stem cells. Its activity decreases with age. Cancer cells can show high telomerase activity. Telomerase is the only DNA polymerase that carries its own primer, a short sequence of RNA. It is a type of reverse transcriptase.

What usually happens when an incorrect base is placed in DNA, or an adduct occurs?

An adduct is an addition to DNA causes by unwanted reactions with base pairs. This causes a 'bulge' in the helices at that point that alerts repair systems.

Nucleotide excision repair is a repair system aimed at fixing bulky changes in a DNA double helix.

If the cell cannot fix the problem, what usually happens?

apoptosis

Are you surprised that these occurrences are more common than mutations? Knowing that mutations create the variety that is so important to evolution, what does that tell you about the enormity of time?

What do thymine dimers result from? What happens, and how is it repaired?

Two adjacent thymines in a DNA strand covalently bond with each other, usually as a result of UV light exposure. The enzyme photolyase recognizes the bulge created and uses visible light to fix the problem.

What is nucleotide excision repair?

Nucleotide excision proteins recognize and excise adducts or errors in the double helix. A short sequence around the error is removed. DNA pol1 then fills the gaps and ligase seals them.

What is base excision repair?

Recognition and removal of a damaged or erroneous base (e.g. a deaminated cytosine) by a glycosylase (e.g. uracil-N-glycosylase)

What does cytosine deaminate to?

In cytosine deamination, cytosine loses it amine and gains a hydroxyl group in its place. This converts its structure to that of uracil.

What does uracil-N-glycosylase do?

The cell recognizes that uracil does not belong in DNA, 'flips' it out of the helix, and clips it off.

What is DNA mismatch repair?

Proofreading during DNA synthesis does not catch every mismatch. Mismatch repair corrects those that remain.

What causes the bulge?

Hydrogen bonds are not paired correctly between the mismatched base pairs.

When there is a mismatch, how does the cell know which one is the wrong base?

In E. coli, methylation is used for mutation recognition. All occurrences of the sequence GTAC are targets of a methylase, which adds a methyl group to each A. DNA replication occurs before methylase activity, so a newly replicated DNA strand is hemimethylated (only the parent strand is methylated). This gives repair systems a window of time in which to recognize bulges and recognize the correct base pairs as those on the strand with a methylated A close by.

When does recombination occur?

prophase I of meiosis

What is the tetrad made of?

4 chromatids, 2 homologous chromosomes

What is synapsis?

When homologous chromosomes pair up in prophase 1 of meiosis; crossing-over takes place at this time

What is chiasma?

The site where crossing over between homologous chromsomes occurs.

What happens when the synaptonemal complex forms?

It helps the crossing over chromosomes swap material downward of the chiasma

Why is this called crossing over?

Because the chromatids crossed over one another and exchanged DNA.

How do you think this contributes to the variety acted on by evolution?

Genetic recombination increases genetic variability.

What does DNA ligase do?

In a circular bacterial chromosome with one replication fork, after replication there is a 'gap' left behind: one phosphodiester bond is missing to completely close the circle. DNA ligase joins the 3' end of the circle with the 5' end by catalyzing this phosphodiester bond. It uses ATP to do this.

On the leading strand, this process only needs to happen once. On the lagging strand, this process must occur wherever Okazaki fragments have been filled in between by DNA pol1.

What are the catalytic activities of DNA pol1?

5' to 3' DNA polymerization activity (to make phosphodiester bonds)

3' to 5' exonuclease activity (to proofread)

5' to 3' exonuclease activity (for removing RNA primers)