Congratulations—you made it to the end of the book. That was not an easy read. If you step back, the story I’ve told you is almost absurd. Cells are made of molecules. Molecules don’t plan, anticipate, or understand. They just move--rapidly and randomly--colliding with each other millions of times per second. There is no conductor. There is no blueprint being consulted as events unfold. No molecule knows what the cell is trying to accomplish. And yet, within this chaotic, res

An active replication fork has two possible fates: it collides with another fork moving toward it or it reaches the end of a chromosome. We'll spend most of this chapter focused on the latter case. But first let's consider what happens when two replication forks meet head-on. Head-to-head encounters The majority of replication forks will run into others moving toward it When this occurs, the cell detects the situation and attaches a ubiquitin molecule to the CMG helicases. Th

Imagine the genome as a massive highway construction project. Thousands of crews are paving road at the same time, each working on its own short stretch. Each crew represents a replication fork. Under normal circumstances, if one crew encounters a problem--a cracked pipe or a large rock in the path of a line--they handle it locally. They pause briefly, clear the obstruction, and continue paving. Cells do something similar. When a replication fork encounters a lesion, local me

Let's reset the scene. A replication fork has stalled. The polymerase cannot move forward due to a lesion that it has reached that hasn't been repaired. If the lesion isn't bypassed by TLS or re-priming in the first 20-30 minutes after RPA-single stranded DNA detection, the fork risks collapsing. The cell's solution to this is surprisingly mechanical: it literally pushes the fork backward. This maneuver, called fork reversal, moves the lesion away from the polymerase and plac

When a polymerase stalls at a lesion, the fork enters a state called fork protection. Within minutes the cell detects RPA-coated single-stranded DNA, stabilizes the replisome, and protects the exposed DNA while it attempts to repair or bypass the lesion. Fork protection is the cell’s first response to a stalled DNA polymerase. The second response, if the lesion isn't successfully bypassed, is fork reversal. This is a more drastic structural rearrangement. Fork reversal reposi

In the previous chapter, I introduced the two major pathways that repair DNA lesions: base excision repair (BER) and nucleotide excision repair (NER). Both operate throughout the genome and throughout the cell cycle. But repair doesn't always occur before a replication fork reaches a lesion. Sometimes a lesion escapes these repair systems and remains in the DNA when S phase begins. When that happens, the replication machinery will eventually encounter it. What happens next de

In addition to DNA polymerase errors, the human genome--even in a perfectly healthy cell--is under intense assault. The threats come from within and without. Endogenous chemicals generated by normal metabolism react with DNA. Exogenous agents from the environment--sunlight, pollutants, cigarette smoke, industrial chemicals--do the same. Together they alter DNA’s chemistry, nick its backbone, and distort its double helical structure. The replication errors we just discussed ar

Surprisingly, the most common DNA polymerase error is not a mismatch at all. It's the insertion of a ribonucleotide--an RNA monomer--into a growing chain instead of a DNA monomer. When that occurs, the bases pair correctly. But the small chemical hydroxyl (-OH) group on the ribose sugar alters the structure of the DNA backbone and makes it more prone to cleavage, increasing the risk of strand breaks. How often does this occur? Recall that the deoxyribonucleotide mismatch erro

DNA replication is astonishingly accurate — but not perfect. Let me quantify that. Before proofreading, replicative polymerases misincorporate roughly once every 100,000 to one million nucleotides. That’s too many. One misincorporation every 100,000 nucleotides would produce tens of thousands of errors in a replicated human genome. In reality, replicated human genomes contain only a handful. Clearly, additional mechanisms must be at work. Cells thus deploy repair pathways to

As the CMG helicase moves along the double helix, propelling the replisome forward and separating the DNA into leading and lagging strands, it creates a physical, or topological, problem. DNA can't be pulled apart without repercussions. The act of unwinding one region of the double helix necessarily affects the regions ahead of it. This is often referred to as the “winding problem.” Let me explain with a human-scale analogy. Imagine two ropes wound tightly around each other l

As I explained in the last chapter, DNA replication has a directional problem. The molecular machines that build DNA--the DNA polymerases--can add new nucleotides in only one direction: 5′ to 3′. That rule is absolute, dictated by the chemistry of the reaction itself. But the two strands of the DNA double helix run in opposite directions. They're antiparallel. So what happens when the replication fork moves forward? One strand can be copied smoothly and continuously. The othe

Let's start off with two indisputable facts. One about DNA. The other about DNA polymerases. Fact one. Recall that the two DNA strands in a double helix are oriented in an antiparallel manner. They run in opposite directions like the lanes of a two-lane highway. Scientists describe strand orientation as either “5’-to-3’” (five prime to three prime) or “3’-to-5’” (three prime to five prime) based on the orientations of the sugars in the strand's backbone. Fact two: In the same

Inside the nucleus of every dividing cell during S phase, massive multi-protein rings race along the DNA. Two of them--both already introduced--are the subject of this chapter. The first one--the CMG helicase--is long and barrel-shaped and has an internal motor. It leads the replisome's charge. Using its motor, it propels itself and the replisome forward, prying the double helix apart into two single strands to be copied. The other--the PCNA sliding clamp--is shaped more like

A human cell enters S phase of the cell cycle. Across its genome, tens of thousands of replication origins have been prepared in advance. Everything is ready but nothing has started yet. Each replication origin has been licensed, or approved for use, based on the presence of two head-to-head MCMs. This is the MCM double hexamer, or MCM-DH. The two MCMs are the inactive precursors of the two helicases that will lead the replication machinery down the DNA, unwinding the double

When the genome is replicated, the cell needs to ensure regions aren't copied multiple times. If that were to happen, it would be like a novel in which random sentences, parts of sentences, chapters and parts of chapters are randomly repeated one or more times. In a genome, such repetitions would cause serious problems. In this chapter, we take a look at the tight regulation involved in making sure that the cell replicates the genome once and only once and with no repeated

If someone had wanted a book in the 15th century, it would have been copied by hand from an existing one. The scribe would have started at page one and gone page by page until the last page was, as they say, “in the books.” A human genome is like a book. So, to copy it, one might imagine the cell would use the same approach: start at one end of each of the 46 chromosomes and progress to the other end. Unfortunately, that wouldn’t work. Human cells must replicate their genom

Having just reviewed all the steps in the cell cycle, we now focus on the fourth step, mitosis, where the duplicated genome is separated into two new cells. I liken this amazing feat of molecular choreography to a line dance. Let's review mitosis from start to finish before covering each step in more detail. Our starting point will be the completion of interphase--that is, phases G1, S, and G2. The genome was copied in S phase. All the chromosomes in the nucleus are now in th

Every day, your body replaces hundreds of billions of cells. Each one must copy over three billion letters of DNA and divide its contents with extraordinary precision. And it must do so at the right time—no sooner, no later. The importance and rate of this process vary dramatically. Some tissues--like bone marrow, the lining of the gut, the skin, and hair follicles--are constantly turning over. Their cells divide continuously to replace those lost to wear. Others divide rarel

Each time a cell divides, the cell copies its entire genome—billions of nucleotides—with extraordinary accuracy. After proofreading and repair, there will be only one error every 10–100 million nucleotides! In this chapter, I review eight reasons why replicating the human genome to that level of accuracy is such a monumental task. The cell overcomes these difficulties, of course. As evidence, consider that on the developmental path from embryo to adult, human cells carry out

Flex your arm. It feels smooth, controlled, almost effortless. But beneath that motion, billions of molecular events are unfolding--tiny proteins pulling, releasing, and resetting in rapid succession. Let's zoom in. Meet myosin, a protein complex in skeletal muscle cells. In this chapter, we'll see how ATP enables molecular-scale myosin movements that cause skeletal muscle contractions. A muscle contraction is not a single event--it’s the result of billions of coordinated mo