DNA and the Gene: Synthesis & Repair

Proteins responsible for opening and stabilizing the double helix

- DNA Helicase - Single stranded DNA binding proteins (SSBPs) - Topoisomerase

Primer

- DNA polymerase requires a primer —which consists of a few nucleotides bonded to the template strand—because it provides a free 3′ hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond.

Key Concepts

- Genes are made of DNA. - When DNA is copied, each strand of a DNA double helix serves as the template for the synthesis of a complementary strand. - DNA synthesis occurs in the 5′ → 3′ direction only and requires a large suite of specialized enzymes. The leading strand is synthesized continuously, but the lagging strand is synthesized as a series of fragments that are then linked together. -Specialized enzymes repair damages to DNA and fix mistakes in DNA synthesis. If these repair enzymes are defective, the mutation rate increases.

Genome

- entire complement of a cell's DNA - genomes are copied before cell division

Replication Bubble

- forms in a chromosome that is actively being replicated. - Replication bubbles grow as DNA replication proceeds, because synthesis is bidirectional.

Xeroderma Pigmentosum (XP)

- rare autosomal recessive disease in humans - individuals with condition are extremely sensitive to ultraviolet light - their skin sunburns and develops rough, scaly patches and irregular dark spots after slight exposure to sunlight

Nucleotide excision repair

- recognizes such types of damage. Its enzymes then remove the single-stranded DNA in the damaged section - DNA can be broken or altered by various chemicals and types of radiation. For example, UV light can cause thymine dimers to form, causing a kink in the DNA strand - The presence of a DNA strand complementary to the damaged strand provides a template for resynthesis of the defective sequences

Okazaki Fragments

- short discontinuous fragments that the lagging strand is synthesized as - DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap - Because Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the discontinuous strand

Telomerase

- the enzyme that adds more repeating bases to the end of the lagging strand, catalyzing the synthesis of DNA from an RNA template that it carries with it - Primase then makes an RNA primer, which DNA polymerase uses to synthesize the lagging strand. Finally, ligase connects the new sequence - This prevents the lagging strand from getting shorter with each replication

DNA Polymerase

- the enzyme that catalyzes DNA synthesis - cleared the way for understanding DNA replication reactions.

Topoisomerase

- the enzyme that cuts and rejoins the DNA downstream of the replication fork, relieving this tension - unwinding the DNA helix creates tension further down the helix

Leading Strand (continuous strand)

- the enzyme's product - it leads into the replication fork and is synthesized continuously in the 5′ --> 3′ direction.

Origin of Replication

- where the replication process beings in bacterial chromosomes Eukaryotes also have bidirectional replication but they have multiple origins of replication and thus have multiple replication bubbles

Hypotheses for how the old and new DNA strands interacted during replication

-Semiconservative replication. -Conservative replication. -Dispersive replication.

Telomere Replication

1.) End is unreplicated 2.) Telomere extends unreplicated end 3.) Telomere repeats activity 4.) Extended single-stranded DNA acts as template

Replicating the ends of linear chromosomes

As the replication fork reaches the end of a linear chromosome, there is no way to replace the RNA primer from the lagging strand with DNA, because there is no available primer for DNA synthesis. The primer is removed, leaving a section of single-stranded DNA (lagging strand) at one end of each new chromosome. This remaining single-stranded DNA is eventually degraded, resulting in shortening of the chromosome.

How is the lagging strand synthesized?

As with the leading strand, synthesis of the lagging strand starts when primase synthesizes a short stretch of RNA that acts as a primer. DNA polymerase III then adds bases to the 3' end of the primer. DNA polymerase moves away from the replication fork, even though helicase continues to open the replication fork and expose single-strand DNA on the lagging strand.

How Does DNA Polymerase Proofread?

DNA polymerase can proofread its work—it checks the match between paired bases, and can correct mismatched bases when they do occur. If the enzyme finds a mismatch, it pauses and removes the mismatched base that was just added. DNA polymerase III can do this because its ε (epsilon) subunit acts as an exonuclease that removes deoxyribonucleotides from DNA. - This proofreading process reduces the error rate to about 1 10^-7.

Deoxyribonucleoside triphosphates (dNTPs)

DNA polymerization is exergonic because the monomers that act as substrates in the reaction are deoxyribonucleoside triphosphates (dNTPs), which have high potential energy because of their three closely packed phosphate groups.

Repairing Mistakes and Damage

DNA replication is very accurate, with an average error rate of less than one mistake per billion bases. DNA polymerase is highly selective in matching complementary bases correctly. As a result, DNA polymerase inserts the incorrect base only about once every 100,000 bases added. If mistakes remain after synthesis is complete or if DNA is damaged, repair enzymes can remove the defective bases and repair them.

How does DNA polymerase proofread? Continued

If—in spite of its proofreading ability—DNA polymerase leaves a mismatched pair behind in the newly synthesized strand, a battery of enzymes springs into action to correct the problem. Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete. Mismatch repair enzymes recognize the mismatched pair, remove a section of the newly synthesized strand that contains the incorrect base, and fill in the correct bases.

The discontinuous replication hypothesis

The discontinuous replication hypothesis stated that once primase synthesizes an RNA primer on the lagging strand, DNA polymerase might synthesize short fragments of DNA along the lagging strand, and that these fragments would later be linked together to form a continuous whole. This hypothesis was tested by Okazaki and his colleagues.

Hershey-Chase Experiment Continued

The radioactive protein was found in the ghosts and the radioactive DNA was found in the cells. The researchers concluded that this result supports that DNA, not protein, is the genetic material. After these results were published, proponents of the protein hypothesis accepted that DNA, not protein, must be the hereditary material. An astonishing claim—that DNA contained all the information for life's complexity—was correct.

Hershey-Chase Experiment

They hypothesized: If genes consist of DNA, then radioactive DNA would be found inside the cells while radioactive proteins would be found only in the ghosts outside the cells. If genes consist of proteins, then only radioactive protein—and no radioactive DNA—would be found inside the cells.

DNA's Secondary Structure

Watson and Crick proposed that two DNA strands line up in the opposite direction to each other, in what is called antiparallel fashion. The antiparallel strands twist to form a double helix. The secondary structure is stabilized by complementary base pairing: Adenine (A) hydrogen bonds with thymine (T). Guanine (G) hydrogen bonds with cytosine (C).

Primase

a type of RNA polymerase, synthesizes a short RNA segment that serves as a primer for DNA synthesis

Single-stranded DNA-binding proteins (SSBPs)

attach to the separated strands to prevent them from closing

DNA ligase

enzyme that joins the okazaki fragments to form a continuous DNA strand

Characteristics of DNA Polymerases

o A critical characteristic of DNA polymerases is that they can only work in one direction. o DNA polymerases can add deoxyribonucleotides to only the 3′ end of a growing DNA chain. As a result, DNA synthesis always proceeds in the 5′ → 3′ direction.

Replication in Somatic cells

o Somatic cells normally lack telomerase. - The chromosomes of somatic cells progressively shorten as the individual ages. o This has led biologists to hypothesize that telomere shortening has a role in limiting the amount of time cells remain in an actively growing state.

DNA's Primary Structure

o The primary structure of DNA has two major components: 1.) A backbone made up of the sugar and phosphate groups of deoxyribonucleotides. 2.) A series of nitrogen-containing bases that project from the backbone. o DNA has directionality—one end has an exposed hydroxyl group on the 3′ carbon of deoxyribose, and the other end has an exposed phosphate group on a 5′ carbon. The molecule thus has a 3′ end and a 5′ end.

What are genes made of?

o While it was known and understood that chromosomes were comprised of DNA and protein, early biologists did not know whether genes were comprised of DNA or protein. o The general consensus at the time supported the hypothesis that genes were comprised of proteins. - This was because of the relative complexity and variability of proteins compared to DNA which is comprised of only four different nucleotides.

Lagging Strand (discontinuous strand)

o it is synthesized discontinuously in the direction away from the replication fork and so lags behind the fork. - This occurs because DNA synthesis must proceed in the 5' 3' direction.

Telomeres

o the regions at the ends of linear chromosomes oReplication of telomeres can be problematic. - While leading-strand synthesis results in a normal copy of the DNA molecule, the telomere on the lagging strand shortens during DNA replication o don't contain genes, but consist of short repeating stretches of bases

Replisome

one large multi-enzyme machine Most of the enzymes responsible for DNA synthesis around the replication fork are joined into one large multi-enzyme machine

Replication Fork

the Y-shaped region where the DNA is split into two separate strands for copying

DNA Helicase

the enzyme that catalyzes the breaking of hydrogen bonds between the two DNA strands to separate them

Capsid

the exterior protein coat of the virus

Ghost

the original parent virus is left behind as a ghost attached to the exterior of the cell

Dispersive Replication

the parent molecule is cut into sections such that the daughter molecules contain old DNA interspersed with newly synthesized DNA

Semiconservative Replication

the parental DNA strands separate and each is used as a template for the synthesis of a new strand. Daughter molecules each consist of one old and one new strand.

Conservative Replication

the parental molecule serves as a template for the synthesis of an entirely new molecule