DNA Replication, Repair and Transcription

Telomerase and telomere replication

Telomeres are repetitive sequences at the ends of linear eukaryotic chromosomes. Conventional DNA polymerases cannot fully copy the 3′ end of a linear template, because removal of the last RNA primer leaves a gap. This is the end-replication problem, and telomerase solves it.

Telomerase is a ribonucleoprotein. Its catalytic subunit, TERT, is a reverse transcriptase, and its integral RNA component, TERC, carries the template that specifies the telomeric repeat. Telomerase therefore synthesises DNA from an RNA template and extends the 3′ overhang by adding repeats. Its activity is that of an RNA-dependent DNA polymerase, which is the same as reverse transcription. It is not a DNA-dependent RNA polymerase, and it is not a 5′→3′ exoribonuclease.

• Reverse transcriptase — an RNA-dependent DNA polymerase that copies RNA into DNA. e.g., Telomerase uses its own RNA template in the same way as a reverse transcriptase.
• DNA-dependent DNA polymerase — copies an existing DNA strand and cannot add telomeric repeats without an RNA template.
• DNA gyrase — a type II topoisomerase that introduces negative supercoils. It is not a telomere-synthesising enzyme.

Chromatin DNA packing ratio

The packing ratio, or fold compaction, is the ratio of the contour length of DNA to the length of the chromatin structure that contains it. The contour length follows from the rise per base pair of B-DNA, which is 0.34 nm. Thus the contour length equals the number of base pairs multiplied by 0.34 nm, and the packing ratio equals the contour length divided by the compacted length. e.g., 1 × 10^6 bp has a contour length of 0.34 mm; if this DNA is packed into 0.1 mm, the packing ratio is 3.4.

At the first level of packing, about 146 to 147 bp of DNA wrap around a histone octamer to form a nucleosome core particle. The DNA makes about 1.65 left-handed superhelical turns around the octamer, and the core particle has a diameter of about 11 nm. To find the DNA wrapped in one nucleosome, multiply the ratio of wrapped DNA length to particle diameter by the diameter to obtain the length in nm, then divide by 0.34 nm per bp.

Eukaryotic pre-mRNA processing

The primary transcript of a eukaryotic gene undergoes three co-transcriptional modifications: 5′ capping, splicing, and 3′ end formation by cleavage and polyadenylation. These steps are coupled to transcription and do not occur in a fixed sequence. Capping and polyadenylation are not prerequisites for splicing.

Splicing takes place by two sequential transesterification reactions. In the first, the 2′-OH group of the branch-point adenosine attacks the 5′ splice site, forming a lariat intermediate. In the second, the 3′-OH of the released exon attacks the 3′ splice site, joining the exons and releasing the lariat intron. Neither step needs an external energy source, although spliceosome assembly uses ATP.

The 3′ end is generated by endonucleolytic cleavage followed by addition of the poly(A) tail. The downstream fragment still attached to RNA polymerase is degraded by the 5′→3′ exonuclease Xrn2, not by a 3′→5′ exonuclease.

Alternative splicing and alternative choice of 3′ end allow one pre-mRNA to yield several mature mRNAs, which can encode different protein isoforms.

The 5′ cap is formed in three enzymatic steps. A triphosphatase removes the γ-phosphate from the 5′-triphosphate end, leaving a 5′-diphosphate. Guanylyltransferase then joins GMP, derived from GTP, through a 5′–5′ triphosphate bridge. Guanine-7-methyltransferase methylates the N-7 position of the terminal guanine to give the m7G cap. Capping begins shortly after the 5′ end of the nascent transcript emerges from the RNA polymerase II exit channel, when it is about 20 to 30 nucleotides long. The donor for the cap is GTP, not ATP.