分子与细胞生物学 11 - L5ab DNA Replication (1)
本期的内容是第五章：DNA复制的前两节内容：实验、复制过程。如果有不太明白的或者有错误的地方随时来找UP主喔~ 文集本部分的参考文献Essential Cell Biology, 5th ed. Alberts, et al. 2019. 部分内容来自khanacademy与维基百科.
本期封面/头图：https://www.bing.com/images/search?view=detailV2&ccid=13TyVcCB&id=0DC14A19ADBF0D18D83EFCC96D336607669A1883&thid=OIP.13TyVcCBsJ91st7lohMMqAHaEK&mediaurl=https%3A%2F%2Fi.ytimg.com%2Fvi%2FcwRzplFt6A8%2Fmaxresdefault.jpg&exph=720&expw=1280&q=DNA+Replication&simid=608036794161496114&ck=EFE3FEF7EF06C6B1B8FAC23BE9061DDD&selectedindex=6&form=IRPRST&ajaxhist=0&vt=0&sim=11
本期内容的笔记以文本的形式呈现。
L5 Replication
5a Replication: Key Experiments
DNA Replication: complementary pairing DNA structure told people something about the replication, but not sufficient
3 models for replication:
Semiconservative model: old strands separate, copy & stay with the new strand
Dispersive model: each strand is composed by short segments of old & new
Conservative model: original strands copy & reassemble, they are conserved
Meselson and Stahl, 1958
To distinguish old DNA from new
Grow bacteria in media (liquid food) with heavy (bacteria grown in 15N (氮15)-containing medium) or light N (bacteria grown in 14N (氮14)-containing medium)
Heavy: bacteria grown incorporate 15N (氮15) in the DNA
Light:bacteria grown incorporate 14N (氮14) in the DNA
How to distinguish molecules that have different weights and densities?
- Density centralization: Put N-DNA into the tubes, then spin.
Experimental Procedures
1) Grow cells for many generations in the heavy medium (15N)
2) Transfer cells to light medium (14N)
3) Grow cells for one or two more replication cycles
4) Isolate DNA
Different Models should give different results - Prediction of the semiconservative model:
This pattern was seen in experiments: third generation: top line 75%, middle line 25%
5b Replication: Copying the strands
Replication - Essential Components
1. Template DNA: DNA that wants to copy
2. dNTPs: deoxynucleoside triphosphate
- As subunits - covalently linking together as a polymer
- As energy - source of energy for the replication reaction
3. DNA Polymerase - enzyme that catalyzes the synthesis the DNA
4. Other proteins
Initiation of DNA replication
- Requires unwinding the double helix
- Start at the Replication Origin (ori) - then local opening of DNA helix
- The replication origin is A-T rich. Because the A-T pairs have fewer H-bonds, easier to separate the strands.
As the replication bubble gets bigger, two replication forks move away from each other; thus, more and more double-stranded DNA unzips.
Eukarytic Chromosome - many origins Replication bubbles will meet together
Elongation
A dNTP base-pairs to the template
- Note: NTP = any Nucleotide Tri-Phosphate, dNTP= the deoxy version (DNA, not RNA)
DNA Polymerase synthesizes by attaching dNTPs onto 3’-OH groups
- Synthesis is 5’ to 3’; the template is read 3’ to 5’.
- High energy bond break (downhill), pyrophosphate (PPi) is released.
DNA synthesis requires energy
- Condensation reaction
- Hydrolyzing nucleotide tri-phosphate
Energy source for replication:
- Deoxynucleotides (ATP is an RNA nucleotide)
- Any base, not just Adenine
Also dATP, dCTP, dGTP, dTTP
- Nucleotide cleaved between 1st and 2nd phosphates
ATP is cleaved between 2nd and 3rd phosphates
DNA polymerase needs a 3’OH to be able to add nucleotides.
RNA Primer - complement to the DNA template to start
dAMP is missing both the 2nd and 3rd phosphate group on the 5' carbon. Normally, the energy released from cleavage of the bonds between phosphates is what allows the addition of a nucleotide to the growing chain. If you can't get energy for DNA synthesis, this uphill condensation reaction cannot occur, and the dAMP nucleotide will not be attached.
ddATP (dideoxy ATP) has 3 phosphates at its 5' end, so one ddATP can be covalently attached to the growing strand. However, ddATP is missing its 3' OH. The DNA polymerase needs the nucleotide at the end of the growing DNA strand to have a 3' OH on which to attach the next nucleotide. Therefore one ddATP will be attached to the growing strand, but replication will cease after that. ddNTPs are called "chain terminators."
Replication bubble
DNA on the leading strand is synthesized from 5’ to 3’, same direction as the fork movement.
DNA on the lagging strand is synthesized in the opposite direction as the fork movement and in discontinuous fragments (Okazaki fragments), from 5’ to 3’.
Lagging strand polymerase repeatedly returns to the fork, and starts synthesizing away from it again.
Rules of replication: 1) always 5’ to 3’, template read 3’ to 5’; 2) start with an RNA primer; 3) two replication forks, four places that replication happen.
本次内容到此结束，感谢阅读！之后的内容将讲述DNA复制过程中的蛋白质与其他活动。
作者：離久-張所長
