2020_Spring_Bis2a_Facciotti_Lecture_21

Learning objectives associated with 2020_Spring_Bis2a_Facciotti_Lecture_21 Describe the challenges of replicating the ends of linear chromosomes. Contrast these challenges with prokaryotic chromosome replication. Create a depiction of the telomerase-mediated telomere-lengthening mechanism that explicitly describes the role of structural RNA in the enzyme complex. Explain how DNA replication is a source of genetic variation, and describe the mechanisms that can change the frequency of mutation and the trade-offs associated with fixing errors.

 

Termination of replication

Telomeres and telomerase

The ends of replication in circular bacterial chromosomes pose few practical problems. However, the ends of linear eukaryotic chromosomes pose a specific problem for DNA replication. Because DNA polymerase can add nucleotides in only one direction (5' to 3'), the leading strand allows for continuous synthesis until the end of the chromosome is reached; however, as the replication complex arrives at the end of the lagging strand there is no place for the primase to "land" and synthesize an RNA primer so that the synthesis of the missing lagging strand DNA fragment at the end of the chromosome can be initiated by the DNA polymerase. Without some mechanism to help fill this gap, this chromosomal end will remain unpaired and will be lost to nucleases. Over time, and several rounds of replication, this would result in the ends of linear chromosomes getting progressively shorter, ultimately compromising the ability of the organism to survive. These ends of the linear chromosomes are known as telomeres, and nearly all eukaryotic species have evolved repetitive sequences that do not code for a specific gene. As a result, these "non-coding" telomeres act as replication buffers and are shortened with each round of DNA replication instead of critical genes. For example, in humans, a six base-pair sequence, TTAGGG, repeats 100 to 1000 times at the end of most chromosomes. Besides acting as a potential buffer, the discovery of the enzyme telomerase helped in understanding how chromosome ends are maintained. Telomerase is an enzyme composed of protein and RNA. Telomerase attaches to the end of the chromosome by complementary base pairing between the RNA component of telomerase and the DNA template. Telomerase uses the RNA as a complementary strand for the short elongation of its complement. The cell repeats this process many times. Once the lagging strand template is sufficiently elongated by telomerase, primase will create a primer followed by DNA polymerase which can now add nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Figure 7. The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine.¹ Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused the extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

 

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Possible NB Discussion Point

Imagine that researchers have invented a drug for humans that upregulates telomerase activity. Would you be excited or would you be skeptical about being part of a clinical trial? Do you trust this drug? Can you think of any negative consequences to activating telomerase to a level that it would not “naturally” be at? Assuming scientists proved without doubt that their drug has no negative side effects -- what are some of the ethical questions you would consider?

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Differences in DNA replication rates between bacteria and eukaryotes

DNA replication has been extremely well studied in bacteria, primarily because of the small size of the genome and the large number of variants available. E. coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that the polymerase adds approximately 1000 nucleotides per second to a growing strand. The process is much more rapid than in eukaryotes. Table 1 summarizes the differences between bacterial and eukaryotic replications.

Table 1. Differences between bacterial and eukaryotic replication.

Differences between prokaryotic and eukaryotic replication
Property	Bacteria	Eukaryotes
Origin of replication	Single	Multiple
Rate of polymerization per polymerase	1000 nucleotides/s	50 to 100 nucleotides/s
Chromosome structure	Circular	Linear
Telomerase	Not present	Present

Link to external resources

Click through a tutorial on DNA replication.

 

Replication design challenge: proofreading

When the cell replicates its DNA, it does so in response to environmental signals that tell the cell it is time to divide. The ideal goal of DNA replication is to produce two identical copies of the double-stranded DNA template and to do it in an amount of time that does not pose an unduly high evolutionarily selective cost. This is a daunting task when you consider that there are ~6,500,000,000 base pairs in the human genome and ~4,500,000 base pairs in the genome of a typical E. coli strain and that Nature has determined that the cells must replicate within 24 hours and 20 minutes, respectively. In either case, many individual biochemical reactions need to take place.

While ideally replication would happen with perfect fidelity, DNA replication, like all other biochemical processes, is imperfect—bases may be left out, extra bases may be added, or bases may be added that do not properly base-pair. In many organisms, many of the mistakes that occur during DNA replication are corrected promptly by DNA polymerase itself via a mechanism known as proofreading. In proofreading, the DNA polymerase "reads" each newly added base via sensing the presence or absence of small structural anomalies before adding the next base to the growing strand. In doing so, a correction can be made.

If the polymerase detects that a newly added base has been paired correctly with the base in the template strand, it adds the next nucleotide. If, however, a wrong nucleotide is added to the growing polymer, the misshaped double helix will cause the DNA polymerase to stall, and it will eject the newly made strand from the polymerizing site. Consequently, the DNA strand will enter an exonuclease site. In this site, DNA polymerase can cleave off the last several nucleotides that were added to the polymer. Once the polymerase removes the incorrect nucleotides, the DNA strand can return to the polymerizing site and new nucleotides will be added again. This proofreading capability comes with some trade-offs: using an error-correcting/more accurate polymerase requires time (the trade-off is speed of replication) and energy (always an important cost to consider). The slower you go, the more accurate you can be. Going too slow, however, may keep you from replicating as fast as your competition, so figuring out the balance is key.

We call errors that are not corrected by proofreading mutations.

Figure 1. Proofreading by DNA polymerase corrects errors during replication.

Replication mistakes and DNA repair

Although DNA replication is typically a highly accurate process and proofreading DNA polymerases helps to keep the error rate low, mistakes still occur. Besides errors of replication, environmental damage may also occur to the DNA. Such uncorrected errors of replication or environmental DNA damage may lead to serious consequences. Therefore, Nature has evolved several mechanisms for repairing damaged or incorrectly synthesized DNA.

Mismatch repair

Errors not corrected during replication may instead get corrected after replication completes; we know this kind of repair as mismatch repairs. Specific enzymes recognize the incorrectly added nucleotide and excise it, replacing it with the correct base. But, how do mismatch repair enzymes recognize which of the two bases is the incorrect one?

In E. coli, after replication, the nitrogenous base adenine gains a methyl group; this means that directly after replication, the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, mismatch repair enzymes can scan the DNA and remove the wrongly incorporated bases from the newly synthesized, non-methylated strand by using the methylated strand as the "correct" template from which to incorporate a new nucleotide. In eukaryotes, the mechanism is not as well understood, but we believe it to involve recognition of unsealed nicks in the new strand, and a short-term, continuing association of some replication proteins with the new daughter strand after replication has completed.

Figure 2. In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

Nucleotide excision repair

Nucleotide excision repair enzymes replace incorrect bases by making a cut on both the 3' and 5' ends of the incorrect base. The entire segment of DNA is removed and replaced with correctly paired nucleotides by the action of a DNA polymerase. Once the bases are filled in, it seal the remaining gap with a phosphodiester linkage catalyzed by the enzyme DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

Figure 3. Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

Consequences of errors in replication, transcription, and translation

Something key to think about:

Cells have evolved a variety of ways to make sure DNA errors are both detected and corrected. We have already discussed several of them. But why did so many mechanisms evolve? From proofreading by the various DNA-dependent DNA polymerases, to the complex repair systems. Such mechanisms did not evolve for errors in transcription or translation. If you are familiar with the processes of transcription and/or translation, think about what the consequences would be of an error in transcription. Would such an error affect the offspring? Would it be lethal to the cell? What about errors in translation? Ask the same questions about the process of translation. What would happen if the wrong amino acid is accidentally put into the growing polypeptide during translation? How do these contrast with DNA replication? If you are not familiar with transcription or translation, don't fret. We'll learn those soon and return to this question again.