EFB325 Cell Physiology

Mechanism of DNA replication and repair

Replication of DNA is semi-conservative

Watson and Crick predicted that DNA replication involved separation of the two strands of DNA, then use of each individual strand as a template for synthesis of a complementary new strand

Matthew Meselson and Franklin Stahl proved this theory in the late '50's using two isotopes of nitrogen, ¹⁴N and ¹⁵N

• grew bacteria in media with ¹⁴N or in media with ¹⁵N, then isolated DNA and used density centrifugation=¹⁵N DNA is more dense, sediments lower in the centrifuge tube
• then grew fresh culture in ¹⁵N, then transferred it to ¹⁴N media long enough for one cell division=DNA sediments as a single band, lighter than ¹⁵N culture
• after two generations on ¹⁴N media, the DNA sediments as two bands=the lower one is the same as after one generation and an upper (lighter) one, same as a culture grown continuously in ¹⁴N
• proves that each strand remains intact through replication and an entirely new strand is made in a duplex with a parent strand at each replication

Replication begins at a specific spot in the middle of a chromosome=origin of replication (ori)

• proteins (called initiator proteins) bind to this region of the chromosome and begin to separate the two strands of DNA (only at the origin)
• this allows the proteins involved in DNA replication to gain access to single-stranded regions of the DNA, which will serve as templates for synthesis of the new strands
• a bacterial chromosome has one origin of replication; eukaryotes have thousands (hundreds on each chromosome)

Separating the DNA strands for replication requires an enzyme

• unwinding the double helix of the DNA throughout the process of replication requires an enzyme=helicase, which breaks the hydrogen bonds holding the strands together (using energy from ATP)
• the single-stranded regions are stabilized and maintained single-stranded by proteins that bind to the DNA=single-strand binding protein (SSB)

DNA synthesis occurs at both strands of a replication fork

• when the initiator protein separates the DNA strands, replication can occur using both strands as templates=replication fork
• as replication proceeds, the replication fork "moves" across the DNA
• the region where the strands were separated is the replication bubble, which has forks at both ends, replication can proceed at both forks, expanding the bubble in both directions
• BUT synthesis of a new strand can occur only in the 5'->3' direction, so on one strand, synthesis is continuous, proceeding from 5'->3'=leading strand
• synthesis using the other strand as a template must be discontinuous, as it proceeds in the direction opposite to the direction in which the replication fork is moving=lagging strand (you could say that synthesis of the new strand in a 5'->3' direction goes backwards)
• the lagging strand is made as a series of small stretches of the new DNA strand=Okazaki fragments, these fragments are later joined together

DNA synthesis is catalyzed by DNA polymerase enzymes

• use deoxyribonucleoside triphosphates (dATP, cCTP, dGTP, and dTTP) as the building blocks
• additional nucleotides are added to the 3' end of the new DNA strand=polymerization involves lengthening the new strand in a 5' to 3' direction (thus it moves from the 3' end to the 5' end of the template strand)
• the proper additional nucleotide to add to the new strand is determined by appropriate base-pairing to the template strand=new strand has a complementary sequence of bases
• DNA polymerase can only start adding on to an existing piece of the new strand (has to start from a region that is already double-stranded, although this can be very short)
• this short piece of the new strand, to which DNA polymerase can start adding new bases=primer
• there are different types of DNA polymerases in the cell, they serve slightly different functions

RNA serves as the primer which DNA polymerase needs to start synthesizing the new strand

• DNA polymerase needs an existing piece of the new strand, so that it can add an additional base at the 3' end=primer
• in DNA replication, the primers that are made are RNA, not DNA (~8-10 bases long)
• the enzyme that makes the primer=primase, starts making a new strand from 5'->3'
• DNA polymerase then takes over and starts adding DNA bases to the primer
• only one primer is needed at the beginning of the leading strand, but for the lagging strand, a primer is needed at the start of each Okazaki fragment
• on the lagging strand, DNA polymerase continues until it approaches the RNA primer of the next Okazaki fragment
• an exonuclease functions to remove the RNA primer, it has activity as a 5'->3' exonuclease
• then the gap is filled in with DNA bases by repair polymerase (by adding on to the 3' end of the previous newly synthesized fragment)
• DNA polymerase cannot connect the 3' end of an Okazaki fragment to the 5' end of the next fragment, there is a single phosphoester bond missing; this bond is formed by the enzyme DNA ligase, using energy from ATP, making the sugar-phosphate backbone of the lagging strand continuous

DNA polymerase can remove bases, as well as adding them=exonuclease activity

• DNA polymerase adds bases from 5'->3', but can also remove bases from the 3' end=exonuclease (an endonuclease cleaves in the middle)
• the 3'->5' exonuclease can remove the last base that was added, if it was added incorrectly=proofreading, then fill in that sequence with the correct base
• proofreading makes replication extremely accurate at producing an exact copy

Telomerase adds sequence onto the ends of eukaryotic chromosomes (to solve the primer dilemma)

• the leading strand can continue synthesis all the way to the end of the template strand, then the DNA polymerase falls off (thus the 3' end of the newly synthesized leading strand goes flush to the end)
• BUT the lagging strand has an RNA primer at the 5' end, which is then removed, leaving a gap; there is no 3' end for DNA polymerase to add bases to in order to fill in that gap at the 5' end of the newly synthesized lagging strand
• after multiple rounds of replication, then the chromosomes would get progressively shorter at each round
• at the ends of eukaryotic chromosomes are stretches of DNA with tandem repeats of the same sequence=telomeres
• the telomere DNA does not have any genes, so cell function is not affected if the telomeres get a little shorter after each cell division (the repeated sequences act like disposable caps on the ends of the chromosome)
• if the telomeres get too short, like in continuously dividing cells (germ cells), then an enzyme called telomerase can bind to the ends of the chromosome and add more DNA onto the 3' end of one of the DNA strands in the telomere, resulting in the newly synthesized complementary strand having a longer telomere sequence also
• the telomeres act like a clock to determine how many cell divisions a cell should go through, telomerase is normally not present in somatic cells, when the chromosomes get too short after a certain number of cell divisions, then the cell dies
• one reason that cancerous cells continue to divide is because they have turned on telomerase, which allows many more cell divisions than a cell would normally be programmed to complete

Changes in the sequence of DNA bases are mutations

• accurate duplication of the DNA sequence in the chromosomes during replication is essential for the cell/organism to survive and reproduce
• changes in the DNA sequence are called mutations

mutations can be caused by:

• incorporation of a mismatched base during replication (failure of DNA polymerase proofreading activity to correct an error)
• chemical reactions that result in the modification of individual bases (either by random hydrolysis reactions, by chemical mutagens, or by radiation, either UV or ionizing radiation)

Errors incorporated by mistakes of DNA replication are corrected quickly

• mismatch repair enzymes recognize a "bulge" in DNA structure caused by the mismatch
• the new strand (with the error) has nicks that are recognized by the repair system and the mismatched base is removed by an exonuclease
• the gap is filled in by a repair DNA polymerase and the last phosphodiester bond formed by DNA ligase

DNA is continually being damaged, must be repaired

• depurination=spontaneous hydrolysis of a A or G base group; can lead to a deletion
• deamination=spontaneous hydrolysis of amino group off of a C, converting it to a U; leads to mutation of G to A after replication
• thymine dimer=covalent linkage of two adjacent T's to each other (caused by UV); leads to a deletion

Cells have enzymes to repair damaged DNA

• the damaged region of the DNA strand is cut out and replaced
• a repair endonuclease enzyme recognizes the damaged base(s), then breaks the phosphodiester bond adjacent to the damaged base (it cuts or nicks the DNA backbone)
• an exonuclease can then remove bases starting at the cut in the backbone
• DNA polymerase then incorporates new bases (which are correctly base-paired) by adding on to the 3' end of the cut DNA strand
• finally, DNA ligase forms the final phosphodiester bond between the last base added by DNA polymerase and the 5' end of the rest of the DNA strand
• organisms that have dysfunctional DNA repair systems accumulate mutations, which eventually lead to cell death
• an early step in the formation of cancer cells is the loss of normal DNA repair functions, allowing cells to accumulate somatic mutations

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